Interleukin-17A Exacerbates Disease Severity in BALB/c MiceSusceptible to Lung Infection with Mycoplasma pulmonis

Maximillion T. Mize,a Xiangle L. Sun,b Jerry W. Simeckaa

aDepartment of Pharmaceutical Sciences and UNT Preclinical Services, University of North Texas SystemCollege of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, USA

bDepartment of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center,Fort Worth, Texas, USA

ABSTRACT Mycoplasmas are atypical bacteria that disrupt the immune response topromote respiratory tract infections and secondary complications. However, not everyimmunologic response that protects or damages the host during mycoplasma infectionis known. Interleukin-17A (IL-17A) is elevated in individuals infected with mycoplasmas,but how IL-17A and its cellular sources dictate disease outcome remains unclear. Here,IL-17A is hypothesized to worsen disease in individuals susceptible to mycoplasma infec-tion. Thus, monoclonal anti-IL-17A antibodies were given to disease-susceptible BALB/cmice and disease-resistant C57BL/6 mice infected with Mycoplasma pulmonis. Neutraliz-ing the function of IL-17A using anti-IL-17A antibodies reduced disease severity dur-ing M. pulmonis infection in BALB/c, but not C57BL/6, mice. Neutralizing IL-17A alsoreduced the incidence of neutrophilic lung lesions during infection in BALB/c mice.Reduced pathology occurred without impacting the bacterial burden, demonstratingthat IL-17A is not required for mycoplasma clearance. The main source of IL-17Athroughout infection in BALB/c mice was CD4� T cells, and neutralizing IL-17A afterinfiltration of the lungs by T cells reduced disease severity, identifying the Th17 re-sponse as a herald of late mycoplasma pathology in susceptible mice. NeutralizingIL-17A did not further reduce disease during M. pulmonis infection in BALB/c micedepleted of neutrophils, suggesting that IL-17A requires the presence of pulmonaryneutrophils to worsen respiratory pathology. IL-17A is a pathological element of mu-rine respiratory mycoplasma infection. Using monoclonal antibodies to neutralize IL-17A could reduce disease severity during mycoplasma infection in humans and do-mesticated animals.

KEYWORDS Th17 cells, bacterial, inflammation, cytokines, mucosa, IL-17,mycoplasma, lung infection

Mycoplasmas are atypical bacteria that cause respiratory and extrapulmonarydiseases in both humans and animals (1–4). Mycoplasma infections in livestock

and poultry have a major economic impact, as they are the causes of respiratory, joint,reproductive, and other infections that reduce the health and productivity of theseanimals. In humans, Mycoplasma pneumoniae is a significant cause of pneumonia, andM. pneumoniae infection exacerbates other respiratory conditions (i.e., asthma andchronic obstructive pulmonary disease [COPD]) (5, 6). Severe infection can be fatalwhen secondary complications (i.e., anemia or encephalitis) arise (7, 8). The worldwideincrease in antibiotic-resistant mycoplasmas is a danger to public health (9–11). Noveltherapies are needed to improve resistance to mycoplasmas and reduce host damageduring infection. Mycoplasmas have many virulence factors that establish infection anddamage neighboring epithelium (12–14). However, host immunity exacerbates diseasepathology, and so, the immune response against mycoplasmas also impacts theoutcome of infection (15–17).

Received 20 April 2018 Returned formodification 22 May 2018 Accepted 29 June2018

Accepted manuscript posted online 9 July2018

Citation Mize MT, Sun XL, Simecka JW. 2018.Interleukin-17A exacerbates disease severityin BALB/c mice susceptible to lung infectionwith Mycoplasma pulmonis. Infect Immun86:e00292-18. https://doi.org/10.1128/IAI.00292-18.

Editor Sabine Ehrt, Weill Cornell MedicalCollege

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Jerry W. Simecka,Jerry.Simecka@unthsc.edu.

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Not every immunologic response that protects or damages the host during respi-ratory mycoplasma infection is completely understood. This blocks the development offully effective vaccines and therapeutic strategies that prevent and treat mycoplasmainfection (11, 18). Infecting mice with Mycoplasma pulmonis, a rodent pathogen thatserves as a model for studying respiratory mycoplasma diseases in its natural host (19,20), has provided insights into mycoplasma diseases, including in humans. Usingmurine infection with M. pulmonis, the importance of lymphocytes in contributing tothe pathology of mycoplasma inflammatory lung disease was demonstrated (21). Forexample, SCID or nude mice (lacking either T and B cells or T cells alone) infected withM. pulmonis display reduced inflammatory damage (22, 23). The depletion of CD4�, butnot CD8�, T cells reduces disease severity in susceptible (i.e., BALB/c) mice and confirmsthat T cells dictate the outcome of infection (24). Different CD4� T helper (Th) subsetspromote contrasting responses during mycoplasma infection (25, 26). In fact, Th2responses contribute to pathology while Th1 responses promote mycoplasma resis-tance (27). Current knowledge of how different T-cell populations contribute to hostprotection and pathology has already produced partially effective mycoplasma vac-cines for animals (28, 29). Continuing to improve our understanding of how different Tcells and their cytokines impact disease outcome is critical for developing fully effectivetherapies that prevent and combat mycoplasma infection.

Interleukin-17A (IL-17A) and Th17 cells increase in children infected with Myco-plasma pneumoniae, and IL-17A is found within the gross lung lesions of cattle infectedwith Mycoplasma mycoides (30, 31), but how IL-17A impacts the immune response tomycoplasma infection is not completely understood. IL-17A is the chief cytokinesecreted by Th17 cells, activating immune pathways involved in infection and disease(32–34). IL-17A enhances host defense during infection by binding to nonhematopoi-etic cells and inducing the production of antimicrobial peptides (i.e., �-defensins andS100 proteins) (35). Blocking IL-17A revealed its role in neutrophil-mediated protectionduring bacterial and fungal infections (36, 37). However, IL-17A activates metallopro-teinases and damaging inflammatory cascades (38, 39). IL-17 production can be initi-ated soon after infection with M. pneumoniae and is dependent upon IL-23 production(40), indicating a potential role in the innate immune response. In vivo neutralization ofIL-23 also resulted in a concomitant reduction in neutrophil recruitment. However,adaptive immune responses may also contribute to IL-17 production, as mice immu-nized with M. pneumoniae extract have increased IL-17 and other inflammatory cyto-kines in the lung after intratracheal inoculation of the extract (41). It appears that IL-17Asecretion can be associated with either innate or adaptive host responses and maycontribute to the inflammatory lesions of severe respiratory mycoplasma infection.However, mycoplasma diseases are multifactorial, and how IL-17A impacts diseaseoutcome could depend upon the genetic background of the host.

In M. pulmonis disease, mouse strains can differ in susceptibility or resistance toinfection and disease (42), and IL-17A may have different impacts upon mycoplasmadiseases depending upon the mouse strain. C57BL/6 mice naturally resist M. pulmonisinfection (43), but susceptibility to disease increases in IL-17RA�/� C57BL/6 mice (44).Other IL-17A homologs also bind IL-17RA (e.g., IL-17C, IL-17E, and IL-17F), and it isunclear whether disease resistance in C57BL/6 mice is specifically associated withIL-17A or another homolog (45–47). Furthermore, it is not known whether a similareffect occurs in BALB/c mice, which are more susceptible to infection with M. pulmonis.The objective here was to examine how IL-17A impacts the response to mycoplasmainfection in disease-susceptible BALB/c mice and disease-resistant C57BL/6 mice. Mono-clonal antibodies that target and neutralize only IL-17A were used to inhibit the effectof the cytokine in infected mice. IL-17A� lung leukocytes were characterized at multipletime points during infection in untreated mice using flow cytometry. As indicatedabove, IL-17 plays a role in the recruitment of neutrophils, but IL-17 can have effects onhost defenses independently of neutrophils (35, 38, 39). To examine whether IL-17Arequires neutrophils to exacerbate M. pulmonis infection, IL-17A was neutralized inneutrophil-depleted BALB/c mice to determine whether IL-17A further reduced the

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severity of disease. Shedding light on the functions of IL-17A during mycoplasmainfection will lead to the development of effective vaccines and therapeutic strategiesthat prevent and treat disease.

RESULTSSerum IL-17A increases in BALB/c, but not C57BL/6, mice infected with M.

pulmonis. To determine if IL-17A is associated with respiratory pathology, disease-susceptible BALB/c mice and disease-resistant C57BL/6 mice were infected with M.pulmonis and sacrificed 14 days later. Cardiac blood was obtained, and serum IL-17Aconcentrations were determined in uninfected and infected mice. Mice were alsoassessed for the characteristics associated with mycoplasma disease.

Serum IL-17A increased in BALB/c, but not C57BL/6, mice infected with M. pulmonis.Prior to infection, serum IL-17A levels in the two strains were equivalent (Fig. 1A). Byday 14 postinfection (p.i.), serum IL-17A remained unchanged in C57BL/6 mice while itincreased over 4-fold in BALB/c mice. Increased serum IL-17A in BALB/c mice wasassociated with mycoplasma pathology characterized by the presence of airway neu-trophils, weight loss, development of gross lung lesions, and persistent infection (Fig.1B to E). These data indicate that increased serum IL-17A is associated with diseaseseverity, suggesting that IL-17A contributes to pathology during severe respiratorymycoplasma infection.

Neutralizing IL-17A daily reduces disease severity during M. pulmonis infectionin BALB/c mice. Increased serum IL-17A in BALB/c, but not C57BL/6, mice infected withM. pulmonis suggests that IL-17A contributes to inflammation and disease in suscep-tible mice. To determine the role of IL-17A in mycoplasma disease, infected BALB/c andC57BL/6 mice were given either polyclonal IgG1 isotype control antibodies or mono-clonal anti-IL-17A antibodies daily. The infected mice were then sacrificed to examinethe impact neutralizing IL-17A had on disease severity and mycoplasma numbers in thelung.

Neutralizing IL-17A reduced disease severity in BALB/c, but not C57BL/6, miceinfected with M. pulmonis. Treating infected BALB/c mice with anti-IL-17A antibodies

FIG 1 Serum IL-17A increased in disease-susceptible BALB/c mice, but not disease-resistant C57BL/6 mice, infectedwith M. pulmonis. BALB/c and C57BL/6 mice were infected with 2 � 105 to 3 � 105 CFU M. pulmonis and sacrificedon day (D) 14 p.i. (A) Cardiac blood was collected, and serum was separated via centrifugation. (B) BAL fluid wasprepared for cytocentrifugation. Slides were air dried, fixed, and stained using a Hema3 kit. Leukocytes weresubsequently characterized based on morphological characteristics. (C) Raw weight. (D) Gross lesions. (E) Bacterialburden, represented as the median log10 CFU per lung. Within-strain comparisons were achieved using aMann-Whitney U test. The data are from one experiment and, unless otherwise stated, represent means andstandard errors of the mean (SEM) (n � 5 mice per time point). *, P � 0.05.

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reduced weight loss, the prevalence of clinical signs, and the incidence of gross lunglesions (Fig. 2A to C). Neutralizing IL-17A failed to change the bacterial burden in bothBALB/c and C57BL/6 mice, suggesting that IL-17A does not contribute to the control ofM. pulmonis in the lungs independently of disease susceptibility (Fig. 2D).

Treating BALB/c mice with anti-IL-17A antibodies daily during M. pulmonis infectionreduced the severity of histologic lung lesions. Examination of individual lung sectionsrevealed that neutralizing IL-17A in infected BALB/c mice reduced airway exudate andalveolar lesions, features characterized by the presence of pulmonary neutrophils (Fig.3) (22, 48). These data suggest that IL-17A contributes to mycoplasma pathology bypromoting neutrophil recruitment into the lungs of susceptible mice. Lung histologyremained unchanged in infected C57BL/6 mice receiving either phosphate-bufferedsaline (PBS) or anti-IL-17A antibodies, suggesting that disease resistance in these miceis not attributable to IL-17A alone (data not shown). Thus, IL-17A does not play identicalroles during the response to mycoplasma infection in BALB/c and C57BL/6 mice.Importantly, IL-17A contributes to histologic lung damage in BALB/c mice susceptibleto infection with M. pulmonis.

To further determine the effect of IL-17A on mycoplasma pathology, weight loss,clinical signs, and gross lung lesions were monitored at multiple time points duringinfection in BALB/c and C57BL/6 mice given either IgG1 antibodies or anti-IL-17Aantibodies daily. Neutralizing IL-17A daily reduced the prevalence of clinical signs byday 7 p.i., lowered weight loss by day 10 p.i., and reduced the incidence of gross lunglesions by day 14 p.i. in BALB/c mice (Fig. 4). Neutralizing IL-17A daily did not changethe prevalence of clinical signs, weight loss, or the development of lung lesions duringM. pulmonis infection in C57BL/6 mice. These data continue to support contrasting roles

FIG 2 Neutralizing IL-17A reduces pathology in BALB/c, but not C57BL/6, mice infected with M. pulmonis.Infected mice were given either polyclonal IgG1 antibodies or monoclonal anti-IL-17A antibodies dailyand sacrificed on day 15 p.i. (A) Weight change, expressed as a percentage, with respect to the initialmass of each individual mouse before infection (BALB/c, n � 24 mice per group; C57BL/6, n � 25 miceper group). (B) Prevalence of clinical signs (BALB/c, n � 24 mice per group; C57BL/6, n � 25 mice pergroup). (C) Gross lesions (BALB/c, n � 24 mice per group; C57BL/6, n � 25 mice per group). (D) Bacterialburden, represented as median log10 CFU per lung (BALB/c, n � 18 mice per group; C57BL/6 control,n � 15 mice; C57BL/6 anti-IL-17A, n � 19 mice). Within-strain comparisons were done using Student’sunpaired t test. All the data are derived from three independent experiments and, unless otherwisestated, represent means and SEM. *, P � 0.05.

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for IL-17A during the response to mycoplasma infection in BALB/c and C57BL/6 mice.These results also suggest that IL-17A plays no role during the initial phase of myco-plasma infection, when controlling bacterial numbers is dependent on innate immunityalone (22, 49). Instead, IL-17A exacerbates pathology during the later stages of infection

FIG 3 Neutralizing IL-17A reduces the severity of neutrophilic lung lesions in BALB/c mice infected with M.pulmonis. Infected BALB/c mice were given either polyclonal IgG1 antibodies or monoclonal anti-IL-17Aantibodies daily and sacrificed on day 15 p.i. The lungs were extracted and prepared for light microscopy. (A)Sections were scored subjectively based on neutrophilic exudate (dashed circle), lymphoid infiltration (arrows),alveolar lesions (dashed square), and epithelial dysplasia (oval). Magnification, �4 (top) and �10 (bottom). (B)Lesion index scores for each category were obtained in a double-blind fashion by the same observer.Comparisons were made using the Mann-Whitney U test. The data are derived from two independentexperiments and represent means and SEM (control, n � 6 mice; anti-IL-17A, n � 7 mice). *, P � 0.05.

FIG 4 IL-17A exacerbates late mycoplasma pathology during infection in BALB/c mice. Infected BALB/c mice (A) and C57BL/6 mice (B) weregiven either PBS or monoclonal anti-IL-17A antibodies daily. Mice were sacrificed at multiple time points to assess weight change, theprevalence of clinical signs, and gross lesions. Comparisons were made relative to uninfected mice using one-way analysis of variance(ANOVA) and a Holm-Sidak post hoc test. The data are derived from three independent experiments and represent means � SEM (n � 15to 20 mice per group). *, P � 0.05.

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in BALB/c mice, coinciding with the activation of adaptive immunity and the appear-ance of T cells in the lung (23).

IL-17A contributes to late mycoplasma pathology during infection in BALB/cmice. The major effect of IL-17A is associated with the adaptive immune response,which begins to participate in disease pathogenesis 5 to 7 days after M. pulmonisinfection in mice (24). To determine exactly when IL-17A plays a role in pathology,infected BALB/c mice were treated with anti-IL-17A antibodies from days �3 to 5 p.i.(early) or days 5 to 14 p.i. (late) (Fig. 5A). Additional groups of infected mice weretreated daily with either PBS or anti-IL-17A antibodies.

Treating BALB/c mice with anti-IL-17A antibodies in specific phases of M. pulmonisinfection had varying impacts on disease severity. Neutralizing IL-17A daily duringinfection in BALB/c mice reduced weight loss and the incidence of gross lesions (Fig. 5Band C). There was no effect on weight loss or gross lesions if infected BALB/c mice weretreated with anti-IL-17A antibodies for the first 5 days p.i. (early). However, BALB/c micetreated from days 5 to 14 p.i. (late) showed a reduction in gross lung lesions similar tothat in mice receiving anti-IL-17A antibodies daily. Despite the effect of anti-IL-17A ongross lesions in “daily” mice and “late” mice, weight loss was no different between“early,” “late,” and control mice. These results demonstrate that IL-17A plays a minorrole in the initial response to infection and instead exacerbates late mycoplasmapathology when disease outcome is dictated by Th responses.

IL-17A is produced primarily by CD4� Th17 cells during M. pulmonis infectionin BALB/c mice. To begin examining when IL-17A is produced during M. pulmonisinfection and identifying the cells responsible, the numbers and types of IL-17A� lungleukocytes from uninfected or infected BALB/c and C57BL/6 mice were determinedusing flow cytometry.

The percentage of IL-17A� lung leukocytes increased in BALB/c, but not C57BL/6,mice infected with M. pulmonis (Fig. 6). The number of IL-17A� lung leukocytesappeared to rise in BALB/c mice by day 3 p.i., while it increased significantly by day 9p.i. (Fig. 7A). IL-17A� CD4� (Th17) and IL-17A� CD8� (Tc17) T cells were the mainsources of IL-17A throughout infection (Fig. 7B). Although lung Th17 cell numbers weredouble those of Tc17 cells at every time point, both populations rose by day 3 p.i. and

FIG 5 Neutralizing IL-17A after adaptive immunity is active reduces mycoplasma pathology. (A) Infectedmice were given either PBS or monoclonal anti-IL-17A antibodies as indicated and then sacrificed on day15 p.i. (B) Weight change, expressed as a percentage with respect to the initial masses of individual miceprior to infection. (C) Gross lesions. Comparisons were made using one-way ANOVA and a Holm-Sidakpost hoc test. The data are derived from three independent experiments and represent means and SEM(control and anti-IL-17A[Daily], n � 19 mice; anti-IL-17A[Early], n � 16 mice; anti-IL-17A[Late], n � 17mice). “a” and “b” represent statistically different populations (P � 0.05).

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reached significantly high numbers by day 9 p.i. Unlike Tc17 cells, however, the numberof Th17 cells remained elevated by day 14 p.i. A population of unknown (CD3�� CD4�

CD8� �� TCR� DX5�) cells and NK cells were secondary sources of IL-17A in the lungsof infected BALB/c mice. Preliminary data suggest that these unknown IL-17A� cellsinclude inflammatory monocytes and neutrophils (data not shown). Few IL-17A� �� Tcells and IL-17A� NKT cells were detected in the lungs throughout infection. Theseresults show that T cells, particularly Th17 cells, are the primary source of IL-17A in the

FIG 6 IL-17A� leukocytes increase in the lungs of BALB/c, but not C57BL/6, mice infected with M.pulmonis. BALB/c and C57BL/6 mice were infected with 2 � 105 to 3 � 105 CFU M. pulmonis and sacrificedon day 14 p.i. Lung leukocytes were isolated, and single-cell suspensions were stained for intracellularIL-17A. Within-strain comparisons were done using a Mann-Whitney U test. The data are from oneexperiment and, unless otherwise stated, represent means and SEM (n � 5 mice per time point). *, P �0.05.

FIG 7 Th17 cells are the primary source of IL-17A in the lungs of infected BALB/c mice. BALB/c mice were sacrificedat various time points, and lung leukocytes were prepared for flow cytometry. (A) Total lung leukocytes and IL-17A�

lung leukocytes. FMO was used on APC/Cy7 to confirm IL-17A secretion by leukocytes. (B) IL-17A� leukocytes werestained for T-cell (CD3�, CD4, CD8, and ��TCR) and NK cell (DX5) surface markers. (Top) CD3�� DX5� lung T cellswere further classified as CD4� CD8� T helper cells, CD4� CD8� cytotoxic T cells, CD4� CD8� DN T cells, and��TCR� �� T cells. (Bottom) Unclassified cells (lacking CD3�, ��TCR, DX5, CD4, and CD8), CD3�� DX5� NK cells, andCD3�� DX5� NKT cells. Significance relative to uninfected controls was analyzed using one-way ANOVA, followedby Dunn’s post hoc test. The data represent means and SEM from two independent experiments (n � 10 mice pertime point). *, P � 0.05.

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lungs of BALB/c mice infected with M. pulmonis. The exacerbation of late mycoplasmapathology by IL-17A is associated with infiltration of the lung by Th17 cells and otherIL-17A� leukocytes.

The exacerbation of mycoplasma pathology by IL-17A depends upon thepresence of neutrophils in BALB/c mice. One major function of IL-17A is recruitingneutrophils to sites of inflammation (50). The production of IL-17A during mycoplasmainfection in susceptible mice is associated with disease pathology, including therecruitment of pulmonary neutrophils (Fig. 1 and 3). To investigate whether the impactof IL-17A on disease outcome depends upon neutrophils, infected BALB/c mice weregiven anti-IL-17A antibodies and/or depleted of neutrophils using anti-Ly6G antibodiesduring infection. Fifteen days after infection, disease severity was assessed. A synergis-tic reduction in disease pathology due to treatment with both anti-IL-17A and anti-Ly6G treatment would indicate that IL-17A and neutrophils exacerbate pathology usingone or more independent mechanisms.

Neutralizing IL-17A or depleting neutrophils reduced the severity of mycoplasmadisease in BALB/c mice (Fig. 8). In infected mice given both anti-IL-17A and anti-Ly6G(Combo), the severity of disease was no different in mice treated with either antibodyalone. Thus, the studies demonstrate that once neutrophils are depleted, there is areduction in disease severity, but concurrent neutralization of IL-17A does not further

FIG 8 The pathology associated with IL-17A is dependent upon the presence of pulmonary neutrophils.Infected mice were given PBS, monoclonal anti-IL-17A antibodies, monoclonal anti-Ly6G antibodies, orboth anti-IL-17A and anti-Ly6G antibodies (Combo) during infection. The mice were sacrificed on day 15p.i., and the lungs were prepared for light microscopy. (A) Weight change. (B) Gross lesions. (C) Lesionindex scores. The data were analyzed using one-way ANOVA followed by Dunn’s post hoc test andrepresent means and SEM from the results of three independent experiments (n � 15 mice per group).“a” and “b” represent statistically different populations (P � 0.05).

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impact disease severity. IL-17A’s role in disease pathogenesis appears to be dependenton neutrophils, which suggests that IL-17A contributes to neutrophil recruitment intothe lungs of susceptible mice during infection.

DISCUSSION

Although T cells and their cytokines play a critical role in modulating immunity tomycoplasmas (23), the role of IL-17A and its cellular sources in the response tomycoplasma infection remains poorly understood. Primarily secreted by Th17 cells,IL-17A plays a role in both host protection and pathology by activating immunedefenses and inflammatory cascades (34, 51). Serum IL-17A and Th17 cells increase inchildren infected with M. pneumoniae, and IL-17A is found within the gross lung lesionsof cattle infected with M. mycoides (30, 31). M. pulmonis is a natural pathogen of miceand provides an opportunity to study normal host-pathogen interactions and re-sponses in mycoplasma infections. Like many infections, the outcome of mycoplasmainfection is influenced by the genetic background of the host (42), and the role ofIL-17A in mycoplasma diseases may similarly be dependent on the host geneticbackground. C57BL/6 mice naturally resist M. pulmonis infection, but disease suscep-tibility increases in IL-17RA�/� C57BL/6 mice (44). Since other IL-17A homologs bindIL-17RA (e.g., IL-17C, IL-17E, and IL-17F), it is unclear whether disease resistance inC57BL/6 mice is associated with IL-17A or another homolog (52). Whether a similareffect occurs in BALB/c mice more susceptible to infection with M. pulmonis is lesscertain due to conflicting reports on the role of IL-17A in mycoplasma disease (40, 41).The objective here was to examine the role, and identify the cellular sources, of IL-17Aduring the response to mycoplasma infection in disease-susceptible BALB/c mice anddisease-resistant C57BL/6 mice.

IL-17A contributes to disease severity during mycoplasma infection in susceptibleBALB/c mice, but not resistant C57BL/6 mice. Serum IL-17A and IL-17A� leukocytesincreased after BALB/c, but not C57BL/6, mice were infected with M. pulmonis, whichsuggests a difference in IL-17A responses between the two mouse strains. In fact,treating BALB/c mice with monoclonal anti-IL-17A antibodies reduced disease severityafter M. pulmonis infection without changing mycoplasma numbers recovered from thelungs. No effect on disease severity or mycoplasma numbers was seen in similarlytreated C57BL/6 mice infected with M. pulmonis. This may not be surprising consideringIL-17A plays contrasting roles during leishmania and mycobacterium infection, provid-ing protection in disease-resistant in C57BL/6 mice while promoting pathology indisease-susceptible BALB/c mice (53). The pathological responses surrounding leish-mania and mycobacterium infections were attributed to IL-17A secretion by Th17 cellsand the resulting recruitment of neutrophils during the later stages of disease inBALB/c, but not C57BL/6, mice (54).

IL-17A plays only a minor role during the initial phase of mycoplasma infection,contributing to disease severity 6 days after infecting BALB/c mice with M. pulmonis.This was confirmed when the severity of gross lung lesions in anti-IL-17A “late” BALB/cmice was no different from that seen in anti-IL-17A “daily” BALB/c mice. It waspreviously shown that this “late” time frame coincides with the activation of adaptiveimmunity and the appearance of T cells in the lung (23). In fact, T cells were the majorsource of IL-17A during mycoplasma infection in BALB/c mice. Although CD4� Th cellsand CD8� Tc cells were major producers of IL-17A, the numbers of Th17 cells weretwice that of Tc17 cells at each time point evaluated. Previous studies have demon-strated that Th cells contribute to inflammatory lung lesions while Tc cells dampenthese damaging responses during mycoplasma infection (24). Thus, Th17 cells likelycontribute to the development of inflammatory lung lesions after M. pulmonis infectionof BALB/c mice.

The production of IL-17A during mycoplasma infection in susceptible mice isassociated with disease pathology, including the recruitment of pulmonary neutrophils.IL-17A plays a significant role in neutrophil-mediated protection during bacterial andfungal infections (36, 37), but secretion of IL-17A by Th17 cells can also exacerbate

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inflammation by recruiting neutrophils into the airways during respiratory diseases (55,56). In susceptible BALB/c mice, depletion of neutrophils reduces disease severity, butconcurrent neutralization of IL-17A does not further impact disease severity. The lack ofan additive effect if mice were treated with both anti-IL-17A and anti-Ly6G antibodiesindicates that the impact of IL-17A on disease severity depends upon the presence ofpulmonary neutrophils. As neutrophils fail to take up mycoplasmas even after in vitrostimulation with IL-17A (57, 58), it appears that neutrophil recruitment does notfacilitate the resolution of mycoplasma infection and instead worsens the inflammatoryresponse in disease-susceptible mice. However, there is support for IL-17A-mediatedneutrophil recruitment promoting resistance to M. pulmonis; increased mycoplasmanumbers in IL-17RA�/� C57BL/6 mice, compared to wild-type mice, suggest thatimpaired IL-17A signaling and neutrophil recruitment can diminish resistance to infec-tion (44). The apparent difference between our results using anti-IL-17A antibodies andthe results of those studies using IL-17RA�/� C57BL/6 mice may be due to other IL-17homologs (e.g., IL-17C, IL-17E, and IL-17F) that also bind IL-17RA (34). Additional studieshave shown that alveolar macrophages in C57BL/6 mice are inherently more effectiveat controlling mycoplasma infection than the alveolar macrophages derived from moresusceptible strains of mice (49, 58). The functions of neutrophils may also differ basedon susceptibility to disease, leading to differences in how IL-17A impacts the outcomeof mycoplasma infection between BALB/c and C57BL/6 mice. Here, IL-17A-mediatedpathology in BALB/c mice is dependent upon neutrophils, but more studies are neededto fully understand how IL-17A and other IL-17 homologs influence the response tomycoplasma infection.

The cytokine response generated against mycoplasmas during infection is likely a majordeterminant of how IL-17A impacts disease outcome. For example, IFN-� or IL-4 signalingworks synergistically with IL-17A to promote contrasting responses (59–62). IL-17A workswith Th2 cytokines to exacerbate leishmania disease in BALB/c mice, while IL-17A and Th1cytokines work synergistically to enhance leishmania resistance in C57BL/6 mice (63, 64). Inmycoplasma disease, IFN-� and IL-4 have differing impacts on the outcome of infection(16). IFN-��/� BALB/c mice develop more severe lung disease and have higher myco-plasma numbers than wild-type mice, but IL-4�/� BALB/c mice have less severe disease andlower mycoplasma numbers (27). Immunizing IL-4�/� mice prior to M. pulmonis infectionresults in more effective resistance to disease, while similar immunization of IFN-��/� micefailed to improve resistance (65). There is no difference in the production of IL-17A betweenwild-type IL-4�/� mice and IFN-��/� mice (17). Although it is not clear what role IL-17Aplays in these knockout mice, it is likely that the different Th responses impact the functionof IL-17A and reveal additional factors that determine the outcome of mycoplasma infec-tion in humans and animals.

This report demonstrates that IL-17A increases disease severity and contributes toneutrophil-mediated lung damage during M. pulmonis infection in disease-susceptibleBALB/c mice, but not disease-resistant C57BL6/mice. These results demonstrate that thefunction of IL-17A in the immune response to mycoplasma can differ based on geneticbackground and susceptibility to disease. Th17 cells are the primary source of IL-17Aduring M. pulmonis infection, and their presence in the lung is associated with theexacerbation of late mycoplasma pathology by IL-17A. Neutrophils are likely recruitedinto the lung in response to IL-17A, and the effect of IL-17A on mycoplasma diseasepathogenesis is dependent upon the presence of neutrophils. Mycoplasmas causeairway inflammation and secondary complications (i.e., anemia and encephalitis) inhumans and animals (1, 2, 6, 66). M. pulmonis is a natural pathogen of mice and can leadto insights into the normal host-pathogen interactions and responses, and these resultsmay reflect those that can occur in other mycoplasma diseases, including those incattle, chickens, and humans. A lack of fully effective therapies or vaccines has under-scored the growing threat mycoplasmas pose to human health and agriculture (11, 18).Neutralizing IL-17A reduces inflammatory damage during infection and disease (67–69).Using monoclonal antibodies to neutralize IL-17A (i.e., secukinumab and ixekizumab) inhumans could serve as a therapy to reduce lung damage during severe mycoplasma

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infection. In addition, developing vaccines that minimize IL-17A responses may alsominimize adverse reactions and improve efficacy, but further studies are needed todetermine the role of IL-17A in resistance to infection. Overall, the production of IL-17Aby Th17 cells contributes to disease severity, highlighting the importance of IL-17A asa potential target for treating severe mycoplasma infection.

MATERIALS AND METHODSMice. Female BALB/cAnHsd and C57BL/6 wild-type mice aged 6 to 8 weeks, tested to be mycoplasma

and virus free, were obtained from Harlan Envigo Laboratory, Inc. The mice were housed in sterilemicroisolator cages supplied with sterile bedding, food, and water, all given ad libitum. Before infectionand sacrifice, we anesthetized the mice with a cocktail of ketamine and xylazine diluted in sterile waterdelivered via intraperitoneal (i.p.) injection. All experiments and protocols were approved by theUniversity of North Texas Health Science Center’s Institutional Animal Care and Use Committee.

Mycoplasmas. The UAB CT strain of M. pulmonis was used in all experiments (70). Stock cultures weregrown as previously described (71). On the day of infection, an aliquot of M. pulmonis stock was dilutedin prewarmed Hayflick’s broth to achieve a final concentration of 2 � 108 to 3 � 108 CFU. Hayflick’smedium was prepared for both broth and agar as follows: ultrapure water (196.25 ml), 1% (wt/vol)phenol red (Sigma-Aldrich Co., Buchs, Switzerland), 5.63 g PPLO broth base (BD Biosciences, San Jose,CA), 0.05 g equine sperm DNA (Sigma-Aldrich Co.), and (for agar only) 2.5 g noble agar (Sigma-Aldrich).Medium was autoclaved on a liquid cycle for 20 min, after which 65 �l Cefobid, 250 mg/ml (Sigma-Aldrich Co.), 2.5 ml Bacto-dextrose, 50% (wt/vol) (BD Biosciences), and 50 ml heat-inactivated equineserum (Thermo-Fisher Scientific, Waltham, MA) were added aseptically. Hayflick’s agar plates wereprepared, and both agar and liquid broth were cooled to room temperature prior to being stored at 4°C.The diluted mycoplasmas were incubated at 37°C for 1 h before we intranasally (i.n.) infected theanesthetized mice with 20 �l of diluted mycoplasmas containing 2 � 105 to 3 � 105 CFU.

Serum. Cardiac blood was collected using a 1-ml syringe, and aliquots were placed in centrifugetubes. The blood was incubated at room temperature for 20 min and subsequently centrifuged at 3,000rpm for 10 min at room temperature. The serum was removed, placed in sterile centrifuge tubes, andprocessed for enzyme-linked immunosorbent assay (ELISA).

IL-17A ELISA. Serum IL-17A was measured by capture ELISA using a BioLegend mouse IL-17A ELISAMax Deluxe kit (BioLegend) according to the manufacturer’s instructions. Briefly, kit-supplied NuncMaxisorp 96-microwell plates were coated overnight at 4°C with capture antibody. The plates werewashed with 0.05% Tween 20 in PBS and blocked using the supplied assay diluent. We washed the platesagain before adding serum and incubating the samples at room temperature with shaking. After anotherwash, detection antibody was added to each well, and the samples were incubated. We washed theplates again, added avidin-horseradish peroxidase (HRP), and allowed the plate to incubate for 1 h. Afteranother wash, we treated the samples with 3,3=,5,5=-tetramethylbenzidine (TMB) substrate solution(BioLegend). The samples were then treated with 2 N H2SO4 before being read using a Synergy HTMulti-Mode Microplate Reader (BioTek, Inc.) at an absorbance of 450 nm. Cytokine levels were deter-mined using Gen5 data analysis software (BioTek, Inc.) by developing a linear regression model tocompare sample values with a standard curve generated from kit-supplied recombinant IL-17A. Thelower and upper limits of detection for IL-17A were 7.8 pg/ml and 500 pg/ml, respectively.

BAL cells. Murine bronchoalveolar lavage (BAL) cells were collected by injecting RPMI 1640 medium(prepared in house using ultrapure water, RPMI 1640 powder [Sigma-Aldrich], 10 mM HEPES [ThermoFisher Scientific], characterized fetal bovine serum [Thermo Fisher Scientific], 100� antibiotics [LifeTechnologies Inc.], 100� L-glutamine [Sigma-Aldrich]) into the tracheas of mice. The tracheal wash wasextracted and centrifuged at 350 � g for 7 min. The cell pellets were resuspended in medium and spunonto glass slides at 800 � g for 5 min using a Cytopro cytocentrifuge (EliTech Group). We stained the BALcells using a Fisher Scientific Hema 3 kit according to the manufacturer’s instructions. The percentagesof macrophages/monocytes, lymphocytes, and neutrophils were determined via light microscopy andused to calculate total cell numbers.

Gross lung lesions. Following sacrifice, we either inflated whole lungs with 2% paraformaldehyde orperfused whole lungs with PBS. We separated each lobe and individually examined them for thepresence of gross lesions. The percent area occupied by lesions on each individual lobe was estimated.This was multiplied by the percentage each lobe contributed to the total mass of the lung, as previouslydescribed (22). The overall gross lesion score for an individual lung was calculated by combining theweighted values generated for each lobe, giving a maximum possible value of 100%.

Bacterial burden. Perfused lungs were placed in GentleMacs C-Tubes containing diluted enzymemixture from the murine lung dissociation kit (Macs Miltenyi Biotec) prepared according to the manu-facturer’s instructions. Lung samples were homogenized for 60 s using a GentleMacs dissociator (MacsMiltenyi Biotec). All homogenization steps used the manufacturer-provided preset for the gentledissociation of murine lungs: Lung_Protocol2. We incubated the lung homogenates in a nutator for 30min at 37°C before homogenizing the samples again for another 60 s. Aliquots of each lung homogenatewere sonicated at 110 amplitudes without pulsing for 60 s. From each sonicated aliquot, we prepared six1:10 serial dilutions in Hayflick’s broth and plated these dilutions onto Hayflick’s agar. Mycoplasmacolonies were counted after 7 days of incubation at 37°C.

Monoclonal antibody treatment. Murine monoclonal anti-IL-17A antibody (BioXCell; clone 1F17)and the IgG1 isotype control antibody (BioXCell; clone MOPC-21) were diluted in sterile-filtered PBS toachieve a final concentration of 0.150 mg/ml. Sterile-filtered PBS served as the control treatment in some

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experiments. We administered anti-IL-17A antibody or the control i.p. in a total volume of 1 ml. The micewere treated daily or at specific stages of disease as outlined in Results.

Murine monoclonal anti-Ly6G antibody (BioXCell; clone 1A8) was diluted in sterile-filtered PBS toachieve a final concentration of 0.300 mg/ml. We also administered anti-Ly6G antibodies i.p. in a totalvolume of 1 ml. Treatment with only anti-Ly6G antibodies occurred every 3 days, beginning day �3 andending day 12 p.i.

Clinical signs. Mice were monitored daily for the following clinical signs of respiratory mycoplasmadisease: (i) matted fur, (ii) elevated heart rate, and (iii) lethargy. Body weights were recorded daily priorto treatment with monoclonal antibodies.

Lung histopathology. Lungs inflated with 2% paraformaldehyde were subsequently fixed inalcohol-formalin (prepared in house with the following formulation: 4% glacial acetic acid [Thermo-Fisher], 6% formaldehyde [Thermo-Fisher], 40% deionized water, and 100% ethanol [Decon Laboratories,Inc.]). Tissues were embedded in paraffin, sectioned at a thickness of 5 �m, and stained with hematoxylinand eosin for light microscopy (by HSRL, Inc.). We scored each lung lobe based on inflammatory damage,as previously described (22). Briefly, sections were scored subjectively by one observer based on thefollowing characteristics of respiratory mycoplasma infection: (i) neutrophil infiltration into the airways(neutrophilic exudate), (ii) peribronchial and perivascular infiltration by lymphocytes (lymphoid infiltra-tion), (iii) infiltration of inflammatory cells into the alveoli (alveolar lesions), and (iv) dysplasia ofrespiratory epithelium (epithelial dysplasia). A score ranging from zero to four was given to each lobe forevery lesion characteristic. For each characteristic, we calculated the index score by dividing the observedvalue by the maximum possible score. The score for each characteristic was then weighted according tothe percentage each lobe contributed to the lung’s total weight; combined, these values provided uswith a total index score for a given characteristic. The maximum possible score for any of the four indiceswas 1.0, representing the highest level of histologic damage.

Lung cell isolation. Perfused lungs were placed in GentleMacs C-Tubes and processed as described in“Bacterial burden” above. Unsonicated lung homogenates were run through a 250-�m mesh filter and dilutedin RPMI 1640 medium. The cells were centrifuged at 800 � g for 10 min at room temperature, and the cellpellets were resuspended in medium. We carefully layered the cells onto Lympholyte-M (Cedarlane) and spunthe samples at 1,200 � g for 20 min at room temperature. Lung leukocytes were isolated by collecting theopaque interphase generated after centrifugation. The collected cells were washed and passed through a70-�m mesh filter to create single-cell suspensions. Lung leukocytes were counted using a Cellometer AutoT4 cell counter (Nexcelom Bioscience).

Flow cytometry. Single-cell suspensions of lung leukocytes were fixed and permeabilized using 2%paraformaldehyde. The fixed cells were washed and then blocked with anti-CD16/anti-CD32 (Fc-block)antibodies (BD Pharmingen) before being stained for 1 h with an isotype control cocktail: fluoresceinisothiocyanate (FITC)/CD3� (Tonbo Biosciences; clone 145.2C11), peridinin chlorophyll protein (PerCP)/CD4 (BioLegend; clone RM4-5), AlexaFluor700/CD8� (BioLegend; clone 53-6.7), phycoerythrin (PE)/��TCR(BD Pharmingen; clone GL3), allophycocyanin (APC)/DX5 (BD Pharmingen; clone DX5), PE-CF594/ROR�t(BD Horizon; clone Q31-378), and APC-Cy7/IL-17A (BioLegend; clone TC11-18H10.1) antibodies. For eachexperiment, fluorescence compensation was achieved using an anti-mouse Ig, compensation particle(BD Biosciences). We identified T cells by gating on all CD3�� leukocytes, while specific T-cell subpopu-lations were differentiated via coexpression of CD3� with CD4 (T helper), CD8 (cytotoxic T cells), ��TCR(�� T cells), or DX5 (NKT cells). CD3�� T cells that did not express CD4 or CD8 or the ��TCR and DX5 cellswere classified as double-negative (DN) T cells. Identification of NK cells required gating on CD3�� DX5�

leukocytes. Leukocytes lacking the five surface molecules analyzed here were classified as unknown. Wesuccessfully identified IL-17A� leukocytes using fluorescence minus one (FMO) on APC-Cy7/IL-17A. Weanalyzed the samples using an LSR II flow cytometer (BD Biosciences), and postacquisition analysis wasperformed using Kaluza analysis software version 1.3 (Beckman Coulter, Inc.). To determine the totalnumber of cells for each leukocyte population, we multiplied the percentages obtained via flowcytometry by the number of cells counted for each sample.

Statistics. Results were analyzed using the appropriate parametric or nonparametric statistical testswhen applicable. The specific statistical methods used for each figure are described in the respectivefigure legends. GraphPad Prism 7 (GraphPad Inc.) software was used, with a P value of �0.05 indicatingstatistical significance.

ACKNOWLEDGMENTSWe acknowledge support of this project through a Robert D. Watkins Fellowship,

kindly provided by the American Society for Microbiology (ASM).We thank the Pre-Clinical Services division at the University of North Texas Health

Science Center (UNTHSC) for providing technical support. We also thank Harlan Jonesfor providing us with his Cytopro Cytocentrifuge. Finally, we thank Leslie Tabor-Simeckaand Calvin Chikelue for all the technical support and advice they provided.

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