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Host Response and Inflammation

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

Maximillion T. Mize, Xiangle L. Sun, Jerry W. Simecka
Sabine Ehrt, Editor
Maximillion T. Mize
aDepartment of Pharmaceutical Sciences and UNT Preclinical Services, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, USA
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  • ORCID record for Maximillion T. Mize
Xiangle L. Sun
bDepartment of Microbiology, Immunology, and Genetics, University of North Texas Health Science Center, Fort Worth, Texas, USA
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Jerry W. Simecka
aDepartment of Pharmaceutical Sciences and UNT Preclinical Services, University of North Texas System College of Pharmacy, University of North Texas Health Science Center, Fort Worth, Texas, USA
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Sabine Ehrt
Weill Cornell Medical College
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DOI: 10.1128/IAI.00292-18
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ABSTRACT

Mycoplasmas are atypical bacteria that disrupt the immune response to promote respiratory tract infections and secondary complications. However, not every immunologic response that protects or damages the host during mycoplasma infection is 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 infection. Thus, monoclonal anti-IL-17A antibodies were given to disease-susceptible BALB/c mice and disease-resistant C57BL/6 mice infected with Mycoplasma pulmonis. Neutralizing the function of IL-17A using anti-IL-17A antibodies reduced disease severity during M. pulmonis infection in BALB/c, but not C57BL/6, mice. Neutralizing IL-17A also reduced the incidence of neutrophilic lung lesions during infection in BALB/c mice. Reduced pathology occurred without impacting the bacterial burden, demonstrating that IL-17A is not required for mycoplasma clearance. The main source of IL-17A throughout infection in BALB/c mice was CD4+ T cells, and neutralizing IL-17A after infiltration of the lungs by T cells reduced disease severity, identifying the Th17 response as a herald of late mycoplasma pathology in susceptible mice. Neutralizing IL-17A did not further reduce disease during M. pulmonis infection in BALB/c mice depleted of neutrophils, suggesting that IL-17A requires the presence of pulmonary neutrophils to worsen respiratory pathology. IL-17A is a pathological element of murine respiratory mycoplasma infection. Using monoclonal antibodies to neutralize IL-17A could reduce disease severity during mycoplasma infection in humans and domesticated animals.

INTRODUCTION

Mycoplasmas are atypical bacteria that cause respiratory and extrapulmonary diseases 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 these animals. In humans, Mycoplasma pneumoniae is a significant cause of pneumonia, and M. pneumoniae infection exacerbates other respiratory conditions (i.e., asthma and chronic obstructive pulmonary disease [COPD]) (5, 6). Severe infection can be fatal when secondary complications (i.e., anemia or encephalitis) arise (7, 8). The worldwide increase in antibiotic-resistant mycoplasmas is a danger to public health (9–11). Novel therapies are needed to improve resistance to mycoplasmas and reduce host damage during infection. Mycoplasmas have many virulence factors that establish infection and damage neighboring epithelium (12–14). However, host immunity exacerbates disease pathology, and so, the immune response against mycoplasmas also impacts the outcome of infection (15–17).

Not every immunologic response that protects or damages the host during respiratory mycoplasma infection is completely understood. This blocks the development of fully effective vaccines and therapeutic strategies that prevent and treat mycoplasma infection (11, 18). Infecting mice with Mycoplasma pulmonis, a rodent pathogen that serves as a model for studying respiratory mycoplasma diseases in its natural host (19, 20), has provided insights into mycoplasma diseases, including in humans. Using murine infection with M. pulmonis, the importance of lymphocytes in contributing to the pathology of mycoplasma inflammatory lung disease was demonstrated (21). For example, SCID or nude mice (lacking either T and B cells or T cells alone) infected with M. pulmonis display reduced inflammatory damage (22, 23). The depletion of CD4+, but not CD8+, T cells reduces disease severity in susceptible (i.e., BALB/c) mice and confirms that T cells dictate the outcome of infection (24). Different CD4+ T helper (Th) subsets promote contrasting responses during mycoplasma infection (25, 26). In fact, Th2 responses contribute to pathology while Th1 responses promote mycoplasma resistance (27). Current knowledge of how different T-cell populations contribute to host protection and pathology has already produced partially effective mycoplasma vaccines for animals (28, 29). Continuing to improve our understanding of how different T cells and their cytokines impact disease outcome is critical for developing fully effective therapies that prevent and combat mycoplasma infection.

Interleukin-17A (IL-17A) and Th17 cells increase in children infected with Mycoplasma pneumoniae, and IL-17A is found within the gross lung lesions of cattle infected with Mycoplasma mycoides (30, 31), but how IL-17A impacts the immune response to mycoplasma infection is not completely understood. IL-17A is the chief cytokine secreted by Th17 cells, activating immune pathways involved in infection and disease (32–34). IL-17A enhances host defense during infection by binding to nonhematopoietic cells and inducing the production of antimicrobial peptides (i.e., β-defensins and S100 proteins) (35). Blocking IL-17A revealed its role in neutrophil-mediated protection during bacterial and fungal infections (36, 37). However, IL-17A activates metalloproteinases and damaging inflammatory cascades (38, 39). IL-17 production can be initiated 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 of IL-23 also resulted in a concomitant reduction in neutrophil recruitment. However, adaptive immune responses may also contribute to IL-17 production, as mice immunized with M. pneumoniae extract have increased IL-17 and other inflammatory cytokines in the lung after intratracheal inoculation of the extract (41). It appears that IL-17A secretion can be associated with either innate or adaptive host responses and may contribute to the inflammatory lesions of severe respiratory mycoplasma infection. However, mycoplasma diseases are multifactorial, and how IL-17A impacts disease outcome could depend upon the genetic background of the host.

In M. pulmonis disease, mouse strains can differ in susceptibility or resistance to infection and disease (42), and IL-17A may have different impacts upon mycoplasma diseases depending upon the mouse strain. C57BL/6 mice naturally resist M. pulmonis infection (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 is unclear whether disease resistance in C57BL/6 mice is specifically associated with IL-17A or another homolog (45–47). Furthermore, it is not known whether a similar effect 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 mycoplasma infection in disease-susceptible BALB/c mice and disease-resistant C57BL/6 mice. Monoclonal antibodies that target and neutralize only IL-17A were used to inhibit the effect of the cytokine in infected mice. IL-17A+ lung leukocytes were characterized at multiple time points during infection in untreated mice using flow cytometry. As indicated above, IL-17 plays a role in the recruitment of neutrophils, but IL-17 can have effects on host defenses independently of neutrophils (35, 38, 39). To examine whether IL-17A requires neutrophils to exacerbate M. pulmonis infection, IL-17A was neutralized in neutrophil-depleted BALB/c mice to determine whether IL-17A further reduced the severity of disease. Shedding light on the functions of IL-17A during mycoplasma infection will lead to the development of effective vaccines and therapeutic strategies that prevent and treat disease.

RESULTS

Serum 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-17A concentrations were determined in uninfected and infected mice. Mice were also assessed 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). By day 14 postinfection (p.i.), serum IL-17A remained unchanged in C57BL/6 mice while it increased over 4-fold in BALB/c mice. Increased serum IL-17A in BALB/c mice was associated with mycoplasma pathology characterized by the presence of airway neutrophils, 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 disease severity, suggesting that IL-17A contributes to pathology during severe respiratory mycoplasma infection.

FIG 1
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FIG 1

Serum IL-17A increased in disease-susceptible BALB/c mice, but not disease-resistant 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 sacrificed on day (D) 14 p.i. (A) Cardiac blood was collected, and serum was separated via centrifugation. (B) BAL fluid was prepared for cytocentrifugation. Slides were air dried, fixed, and stained using a Hema3 kit. Leukocytes were subsequently characterized based on morphological characteristics. (C) Raw weight. (D) Gross lesions. (E) Bacterial burden, represented as the median log10 CFU per lung. Within-strain comparisons were achieved using a Mann-Whitney U test. The data are from one experiment and, unless otherwise stated, represent means and standard errors of the mean (SEM) (n = 5 mice per time point). *, P < 0.05.

Neutralizing IL-17A daily reduces disease severity during M. pulmonis infection in BALB/c mice.Increased serum IL-17A in BALB/c, but not C57BL/6, mice infected with M. pulmonis suggests that IL-17A contributes to inflammation and disease in susceptible mice. To determine the role of IL-17A in mycoplasma disease, infected BALB/c and C57BL/6 mice were given either polyclonal IgG1 isotype control antibodies or monoclonal anti-IL-17A antibodies daily. The infected mice were then sacrificed to examine the impact neutralizing IL-17A had on disease severity and mycoplasma numbers in the lung.

Neutralizing IL-17A reduced disease severity in BALB/c, but not C57BL/6, mice infected with M. pulmonis. Treating infected BALB/c mice with anti-IL-17A antibodies reduced weight loss, the prevalence of clinical signs, and the incidence of gross lung lesions (Fig. 2A to C). Neutralizing IL-17A failed to change the bacterial burden in both BALB/c and C57BL/6 mice, suggesting that IL-17A does not contribute to the control of M. pulmonis in the lungs independently of disease susceptibility (Fig. 2D).

FIG 2
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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 daily and sacrificed on day 15 p.i. (A) Weight change, expressed as a percentage, with respect to the initial mass of each individual mouse before infection (BALB/c, n = 24 mice per group; C57BL/6, n = 25 mice per group). (B) Prevalence of clinical signs (BALB/c, n = 24 mice per group; C57BL/6, n = 25 mice per group). (C) Gross lesions (BALB/c, n = 24 mice per group; C57BL/6, n = 25 mice per group). (D) Bacterial burden, 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's unpaired t test. All the data are derived from three independent experiments and, unless otherwise stated, represent means and SEM. *, P < 0.05.

Treating BALB/c mice with anti-IL-17A antibodies daily during M. pulmonis infection reduced the severity of histologic lung lesions. Examination of individual lung sections revealed that neutralizing IL-17A in infected BALB/c mice reduced airway exudate and alveolar lesions, features characterized by the presence of pulmonary neutrophils (Fig. 3) (22, 48). These data suggest that IL-17A contributes to mycoplasma pathology by promoting neutrophil recruitment into the lungs of susceptible mice. Lung histology remained unchanged in infected C57BL/6 mice receiving either phosphate-buffered saline (PBS) or anti-IL-17A antibodies, suggesting that disease resistance in these mice is not attributable to IL-17A alone (data not shown). Thus, IL-17A does not play identical roles 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 susceptible to infection with M. pulmonis.

FIG 3
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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-17A antibodies 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 independent experiments and represent means and SEM (control, n = 6 mice; anti-IL-17A, n = 7 mice). *, P < 0.05.

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 during infection in BALB/c and C57BL/6 mice given either IgG1 antibodies or anti-IL-17A antibodies daily. Neutralizing IL-17A daily reduced the prevalence of clinical signs by day 7 p.i., lowered weight loss by day 10 p.i., and reduced the incidence of gross lung lesions by day 14 p.i. in BALB/c mice (Fig. 4). Neutralizing IL-17A daily did not change the prevalence of clinical signs, weight loss, or the development of lung lesions during M. pulmonis infection in C57BL/6 mice. These data continue to support contrasting roles 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 mycoplasma infection, when controlling bacterial numbers is dependent on innate immunity alone (22, 49). Instead, IL-17A exacerbates pathology during the later stages of infection in BALB/c mice, coinciding with the activation of adaptive immunity and the appearance of T cells in the lung (23).

FIG 4
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FIG 4

IL-17A exacerbates late mycoplasma pathology during infection in BALB/c mice. Infected BALB/c mice (A) and C57BL/6 mice (B) were given either PBS or monoclonal anti-IL-17A antibodies daily. Mice were sacrificed at multiple time points to assess weight change, the prevalence 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 = 15 to 20 mice per group). *, P ≤ 0.05.

IL-17A contributes to late mycoplasma pathology during infection in BALB/c mice.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. pulmonis infection 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 were treated daily with either PBS or anti-IL-17A antibodies.

FIG 5
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FIG 5

Neutralizing IL-17A after adaptive immunity is active reduces mycoplasma pathology. (A) Infected mice were given either PBS or monoclonal anti-IL-17A antibodies as indicated and then sacrificed on day 15 p.i. (B) Weight change, expressed as a percentage with respect to the initial masses of individual mice prior to infection. (C) Gross lesions. Comparisons were made using one-way ANOVA and a Holm-Sidak post 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 = 17 mice). “a” and “b” represent statistically different populations (P < 0.05).

Treating BALB/c mice with anti-IL-17A antibodies in specific phases of M. pulmonis infection had varying impacts on disease severity. Neutralizing IL-17A daily during infection in BALB/c mice reduced weight loss and the incidence of gross lesions (Fig. 5B and C). There was no effect on weight loss or gross lesions if infected BALB/c mice were treated with anti-IL-17A antibodies for the first 5 days p.i. (early). However, BALB/c mice treated from days 5 to 14 p.i. (late) showed a reduction in gross lung lesions similar to that in mice receiving anti-IL-17A antibodies daily. Despite the effect of anti-IL-17A on gross 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 minor role in the initial response to infection and instead exacerbates late mycoplasma pathology when disease outcome is dictated by Th responses.

IL-17A is produced primarily by CD4+ Th17 cells during M. pulmonis infection in BALB/c mice.To begin examining when IL-17A is produced during M. pulmonis infection and identifying the cells responsible, the numbers and types of IL-17A+ lung leukocytes from uninfected or infected BALB/c and C57BL/6 mice were determined using 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 leukocytes appeared to rise in BALB/c mice by day 3 p.i., while it increased significantly by day 9 p.i. (Fig. 7A). IL-17A+ CD4+ (Th17) and IL-17A+ CD8+ (Tc17) T cells were the main sources of IL-17A throughout infection (Fig. 7B). Although lung Th17 cell numbers were double those of Tc17 cells at every time point, both populations rose by day 3 p.i. and reached significantly high numbers by day 9 p.i. Unlike Tc17 cells, however, the number of Th17 cells remained elevated by day 14 p.i. A population of unknown (CD3ε− CD4− CD8− γδ TCR− DX5−) cells and NK cells were secondary sources of IL-17A in the lungs of infected BALB/c mice. Preliminary data suggest that these unknown IL-17A+ cells include inflammatory monocytes and neutrophils (data not shown). Few IL-17A+ γδ T cells and IL-17A+ NKT cells were detected in the lungs throughout infection. These results show that T cells, particularly Th17 cells, are the primary source of IL-17A in the lungs of BALB/c mice infected with M. pulmonis. The exacerbation of late mycoplasma pathology by IL-17A is associated with infiltration of the lung by Th17 cells and other IL-17A+ leukocytes.

FIG 6
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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 sacrificed on day 14 p.i. Lung leukocytes were isolated, and single-cell suspensions were stained for intracellular IL-17A. Within-strain comparisons were done using a Mann-Whitney U test. The data are from one experiment and, unless otherwise stated, represent means and SEM (n = 5 mice per time point). *, P < 0.05.

FIG 7
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FIG 7

Th17 cells are the primary source of IL-17A in the lungs of infected BALB/c mice. BALB/c mice were sacrificed at 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 were stained for T-cell (CD3ε, CD4, CD8, and γδTCR) and NK cell (DX5) surface markers. (Top) CD3ε+ DX5− lung T cells were further classified as CD4+ CD8− T helper cells, CD4− CD8+ cytotoxic T cells, CD4− CD8− DN T cells, and γδTCR+ γδ T cells. (Bottom) Unclassified cells (lacking CD3ε, γδTCR, DX5, CD4, and CD8), CD3ε− DX5+ NK cells, and CD3ε+ DX5+ NKT cells. Significance relative to uninfected controls was analyzed using one-way ANOVA, followed by Dunn's post hoc test. The data represent means and SEM from two independent experiments (n = 10 mice per time point). *, P < 0.05.

The exacerbation of mycoplasma pathology by IL-17A depends upon the presence of neutrophils in BALB/c mice.One major function of IL-17A is recruiting neutrophils to sites of inflammation (50). The production of IL-17A during mycoplasma infection in susceptible mice is associated with disease pathology, including the recruitment of pulmonary neutrophils (Fig. 1 and 3). To investigate whether the impact of IL-17A on disease outcome depends upon neutrophils, infected BALB/c mice were given anti-IL-17A antibodies and/or depleted of neutrophils using anti-Ly6G antibodies during infection. Fifteen days after infection, disease severity was assessed. A synergistic 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 using one or more independent mechanisms.

Neutralizing IL-17A or depleting neutrophils reduced the severity of mycoplasma disease 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 antibody alone. Thus, the studies demonstrate that once neutrophils are depleted, there is a reduction in disease severity, but concurrent neutralization of IL-17A does not further impact disease severity. IL-17A's role in disease pathogenesis appears to be dependent on neutrophils, which suggests that IL-17A contributes to neutrophil recruitment into the lungs of susceptible mice during infection.

FIG 8
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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, or both anti-IL-17A and anti-Ly6G antibodies (Combo) during infection. The mice were sacrificed on day 15 p.i., and the lungs were prepared for light microscopy. (A) Weight change. (B) Gross lesions. (C) Lesion index scores. The data were analyzed using one-way ANOVA followed by Dunn's post hoc test and represent 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).

DISCUSSION

Although T cells and their cytokines play a critical role in modulating immunity to mycoplasmas (23), the role of IL-17A and its cellular sources in the response to mycoplasma infection remains poorly understood. Primarily secreted by Th17 cells, IL-17A plays a role in both host protection and pathology by activating immune defenses and inflammatory cascades (34, 51). Serum IL-17A and Th17 cells increase in children infected with M. pneumoniae, and IL-17A is found within the gross lung lesions of cattle infected with M. mycoides (30, 31). M. pulmonis is a natural pathogen of mice and provides an opportunity to study normal host-pathogen interactions and responses in mycoplasma infections. Like many infections, the outcome of mycoplasma infection is influenced by the genetic background of the host (42), and the role of IL-17A in mycoplasma diseases may similarly be dependent on the host genetic background. C57BL/6 mice naturally resist M. pulmonis infection, but disease susceptibility increases in IL-17RA−/− C57BL/6 mice (44). Since other IL-17A homologs bind IL-17RA (e.g., IL-17C, IL-17E, and IL-17F), it is unclear whether disease resistance in C57BL/6 mice is associated with IL-17A or another homolog (52). Whether a similar effect occurs in BALB/c mice more susceptible to infection with M. pulmonis is less certain 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-17A during the response to mycoplasma infection in disease-susceptible BALB/c mice and disease-resistant C57BL/6 mice.

IL-17A contributes to disease severity during mycoplasma infection in susceptible BALB/c mice, but not resistant C57BL/6 mice. Serum IL-17A and IL-17A+ leukocytes increased after BALB/c, but not C57BL/6, mice were infected with M. pulmonis, which suggests 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 severity after M. pulmonis infection without changing mycoplasma numbers recovered from the lungs. No effect on disease severity or mycoplasma numbers was seen in similarly treated C57BL/6 mice infected with M. pulmonis. This may not be surprising considering IL-17A plays contrasting roles during leishmania and mycobacterium infection, providing protection in disease-resistant in C57BL/6 mice while promoting pathology in disease-susceptible BALB/c mice (53). The pathological responses surrounding leishmania and mycobacterium infections were attributed to IL-17A secretion by Th17 cells and the resulting recruitment of neutrophils during the later stages of disease in BALB/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/c mice was no different from that seen in anti-IL-17A “daily” BALB/c mice. It was previously shown that this “late” time frame coincides with the activation of adaptive immunity and the appearance of T cells in the lung (23). In fact, T cells were the major source of IL-17A during mycoplasma infection in BALB/c mice. Although CD4+ Th cells and CD8+ Tc cells were major producers of IL-17A, the numbers of Th17 cells were twice that of Tc17 cells at each time point evaluated. Previous studies have demonstrated that Th cells contribute to inflammatory lung lesions while Tc cells dampen these damaging responses during mycoplasma infection (24). Thus, Th17 cells likely contribute to the development of inflammatory lung lesions after M. pulmonis infection of BALB/c mice.

The production of IL-17A during mycoplasma infection in susceptible mice is associated with disease pathology, including the recruitment of pulmonary neutrophils. IL-17A plays a significant role in neutrophil-mediated protection during bacterial and fungal infections (36, 37), but secretion of IL-17A by Th17 cells can also exacerbate inflammation by recruiting neutrophils into the airways during respiratory diseases (55, 56). In susceptible BALB/c mice, depletion of neutrophils reduces disease severity, but concurrent neutralization of IL-17A does not further impact disease severity. The lack of an additive effect if mice were treated with both anti-IL-17A and anti-Ly6G antibodies indicates that the impact of IL-17A on disease severity depends upon the presence of pulmonary neutrophils. As neutrophils fail to take up mycoplasmas even after in vitro stimulation with IL-17A (57, 58), it appears that neutrophil recruitment does not facilitate the resolution of mycoplasma infection and instead worsens the inflammatory response in disease-susceptible mice. However, there is support for IL-17A-mediated neutrophil recruitment promoting resistance to M. pulmonis; increased mycoplasma numbers in IL-17RA−/− C57BL/6 mice, compared to wild-type mice, suggest that impaired IL-17A signaling and neutrophil recruitment can diminish resistance to infection (44). The apparent difference between our results using anti-IL-17A antibodies and the results of those studies using IL-17RA−/− C57BL/6 mice may be due to other IL-17 homologs (e.g., IL-17C, IL-17E, and IL-17F) that also bind IL-17RA (34). Additional studies have shown that alveolar macrophages in C57BL/6 mice are inherently more effective at controlling mycoplasma infection than the alveolar macrophages derived from more susceptible strains of mice (49, 58). The functions of neutrophils may also differ based on susceptibility to disease, leading to differences in how IL-17A impacts the outcome of mycoplasma infection between BALB/c and C57BL/6 mice. Here, IL-17A-mediated pathology in BALB/c mice is dependent upon neutrophils, but more studies are needed to fully understand how IL-17A and other IL-17 homologs influence the response to mycoplasma infection.

The cytokine response generated against mycoplasmas during infection is likely a major determinant of how IL-17A impacts disease outcome. For example, IFN-γ or IL-4 signaling works synergistically with IL-17A to promote contrasting responses (59–62). IL-17A works with Th2 cytokines to exacerbate leishmania disease in BALB/c mice, while IL-17A and Th1 cytokines work synergistically to enhance leishmania resistance in C57BL/6 mice (63, 64). In mycoplasma disease, IFN-γ and IL-4 have differing impacts on the outcome of infection (16). IFN-γ−/− BALB/c mice develop more severe lung disease and have higher mycoplasma numbers than wild-type mice, but IL-4−/− BALB/c mice have less severe disease and lower mycoplasma numbers (27). Immunizing IL-4−/− mice prior to M. pulmonis infection results in more effective resistance to disease, while similar immunization of IFN-γ−/− mice failed to improve resistance (65). There is no difference in the production of IL-17A between wild-type IL-4−/− mice and IFN-γ−/− mice (17). Although it is not clear what role IL-17A plays in these knockout mice, it is likely that the different Th responses impact the function of IL-17A and reveal additional factors that determine the outcome of mycoplasma infection in humans and animals.

This report demonstrates that IL-17A increases disease severity and contributes to neutrophil-mediated lung damage during M. pulmonis infection in disease-susceptible BALB/c mice, but not disease-resistant C57BL6/mice. These results demonstrate that the function of IL-17A in the immune response to mycoplasma can differ based on genetic background and susceptibility to disease. Th17 cells are the primary source of IL-17A during M. pulmonis infection, and their presence in the lung is associated with the exacerbation of late mycoplasma pathology by IL-17A. Neutrophils are likely recruited into the lung in response to IL-17A, and the effect of IL-17A on mycoplasma disease pathogenesis is dependent upon the presence of neutrophils. Mycoplasmas cause airway inflammation and secondary complications (i.e., anemia and encephalitis) in humans and animals (1, 2, 6, 66). M. pulmonis is a natural pathogen of mice and can lead to insights into the normal host-pathogen interactions and responses, and these results may reflect those that can occur in other mycoplasma diseases, including those in cattle, chickens, and humans. A lack of fully effective therapies or vaccines has underscored 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) in humans could serve as a therapy to reduce lung damage during severe mycoplasma infection. In addition, developing vaccines that minimize IL-17A responses may also minimize adverse reactions and improve efficacy, but further studies are needed to determine the role of IL-17A in resistance to infection. Overall, the production of IL-17A by Th17 cells contributes to disease severity, highlighting the importance of IL-17A as a potential target for treating severe mycoplasma infection.

MATERIALS AND METHODS

Mice.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 sterile microisolator cages supplied with sterile bedding, food, and water, all given ad libitum. Before infection and sacrifice, we anesthetized the mice with a cocktail of ketamine and xylazine diluted in sterile water delivered via intraperitoneal (i.p.) injection. All experiments and protocols were approved by the University 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 were grown as previously described (71). On the day of infection, an aliquot of M. pulmonis stock was diluted in prewarmed Hayflick's broth to achieve a final concentration of 2 × 108 to 3 × 108 CFU. Hayflick’s medium 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 μl Cefobid, 250 mg/ml (Sigma-Aldrich Co.), 2.5 ml Bacto-dextrose, 50% (wt/vol) (BD Biosciences), and 50 ml heat-inactivated equine serum (Thermo-Fisher Scientific, Waltham, MA) were added aseptically. Hayflick’s agar plates were prepared, 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 the anesthetized mice with 20 μl 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 centrifuge tubes. The blood was incubated at room temperature for 20 min and subsequently centrifuged at 3,000 rpm for 10 min at room temperature. The serum was removed, placed in sterile centrifuge tubes, and processed for enzyme-linked immunosorbent assay (ELISA).

IL-17A ELISA.Serum IL-17A was measured by capture ELISA using a BioLegend mouse IL-17A ELISA Max Deluxe kit (BioLegend) according to the manufacturer's instructions. Briefly, kit-supplied Nunc Maxisorp 96-microwell plates were coated overnight at 4°C with capture antibody. The plates were washed with 0.05% Tween 20 in PBS and blocked using the supplied assay diluent. We washed the plates again before adding serum and incubating the samples at room temperature with shaking. After another wash, detection antibody was added to each well, and the samples were incubated. We washed the plates again, added avidin-horseradish peroxidase (HRP), and allowed the plate to incubate for 1 h. After another 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 HT Multi-Mode Microplate Reader (BioTek, Inc.) at an absorbance of 450 nm. Cytokine levels were determined using Gen5 data analysis software (BioTek, Inc.) by developing a linear regression model to compare sample values with a standard curve generated from kit-supplied recombinant IL-17A. The lower 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 [Thermo Fisher Scientific], characterized fetal bovine serum [Thermo Fisher Scientific], 100× antibiotics [Life Technologies Inc.], 100× l-glutamine [Sigma-Aldrich]) into the tracheas of mice. The tracheal wash was extracted and centrifuged at 350 × g for 7 min. The cell pellets were resuspended in medium and spun onto glass slides at 800 × g for 5 min using a Cytopro cytocentrifuge (EliTech Group). We stained the BAL cells using a Fisher Scientific Hema 3 kit according to the manufacturer's instructions. The percentages of macrophages/monocytes, lymphocytes, and neutrophils were determined via light microscopy and used to calculate total cell numbers.

Gross lung lesions.Following sacrifice, we either inflated whole lungs with 2% paraformaldehyde or perfused whole lungs with PBS. We separated each lobe and individually examined them for the presence 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 previously described (22). The overall gross lesion score for an individual lung was calculated by combining the weighted values generated for each lobe, giving a maximum possible value of 100%.

Bacterial burden.Perfused lungs were placed in GentleMacs C-Tubes containing diluted enzyme mixture from the murine lung dissociation kit (Macs Miltenyi Biotec) prepared according to the manufacturer's instructions. Lung samples were homogenized for 60 s using a GentleMacs dissociator (Macs Miltenyi Biotec). All homogenization steps used the manufacturer-provided preset for the gentle dissociation of murine lungs: Lung_Protocol2. We incubated the lung homogenates in a nutator for 30 min at 37°C before homogenizing the samples again for another 60 s. Aliquots of each lung homogenate were sonicated at 110 amplitudes without pulsing for 60 s. From each sonicated aliquot, we prepared six 1:10 serial dilutions in Hayflick's broth and plated these dilutions onto Hayflick's agar. Mycoplasma colonies 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 to achieve a final concentration of 0.150 mg/ml. Sterile-filtered PBS served as the control treatment in some experiments. We administered anti-IL-17A antibody or the control i.p. in a total volume of 1 ml. The mice were 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 to achieve a final concentration of 0.300 mg/ml. We also administered anti-Ly6G antibodies i.p. in a total volume of 1 ml. Treatment with only anti-Ly6G antibodies occurred every 3 days, beginning day −3 and ending day 12 p.i.

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

Lung histopathology.Lungs inflated with 2% paraformaldehyde were subsequently fixed in alcohol-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 μm, and stained with hematoxylin and 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 the following characteristics of respiratory mycoplasma infection: (i) neutrophil infiltration into the airways (neutrophilic exudate), (ii) peribronchial and perivascular infiltration by lymphocytes (lymphoid infiltration), (iii) infiltration of inflammatory cells into the alveoli (alveolar lesions), and (iv) dysplasia of respiratory epithelium (epithelial dysplasia). A score ranging from zero to four was given to each lobe for every lesion characteristic. For each characteristic, we calculated the index score by dividing the observed value by the maximum possible score. The score for each characteristic was then weighted according to the percentage each lobe contributed to the lung's total weight; combined, these values provided us with a total index score for a given characteristic. The maximum possible score for any of the four indices was 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-μm mesh filter and diluted in RPMI 1640 medium. The cells were centrifuged at 800 × g for 10 min at room temperature, and the cell pellets were resuspended in medium. We carefully layered the cells onto Lympholyte-M (Cedarlane) and spun the samples at 1,200 × g for 20 min at room temperature. Lung leukocytes were isolated by collecting the opaque interphase generated after centrifugation. The collected cells were washed and passed through a 70-μm mesh filter to create single-cell suspensions. Lung leukocytes were counted using a Cellometer Auto T4 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: fluorescein isothiocyanate (FITC)/CD3ε (Tonbo Biosciences; clone 145.2C11), peridinin chlorophyll protein (PerCP)/CD4 (BioLegend; clone RM4-5), AlexaFluor700/CD8α (BioLegend; clone 53-6.7), phycoerythrin (PE)/γδTCR (BD Pharmingen; clone GL3), allophycocyanin (APC)/DX5 (BD Pharmingen; clone DX5), PE-CF594/RORγt (BD Horizon; clone Q31-378), and APC-Cy7/IL-17A (BioLegend; clone TC11-18H10.1) antibodies. For each experiment, fluorescence compensation was achieved using an anti-mouse Ig, κ compensation particle (BD Biosciences). We identified T cells by gating on all CD3ε+ leukocytes, while specific T-cell subpopulations were differentiated via coexpression of CD3ε with CD4 (T helper), CD8 (cytotoxic T cells), γδTCR (γδ T cells), or DX5 (NKT cells). CD3ε+ T cells that did not express CD4 or CD8 or the γδTCR and DX5 cells were classified as double-negative (DN) T cells. Identification of NK cells required gating on CD3ε− DX5+ leukocytes. Leukocytes lacking the five surface molecules analyzed here were classified as unknown. We successfully identified IL-17A+ leukocytes using fluorescence minus one (FMO) on APC-Cy7/IL-17A. We analyzed the samples using an LSR II flow cytometer (BD Biosciences), and postacquisition analysis was performed using Kaluza analysis software version 1.3 (Beckman Coulter, Inc.). To determine the total number of cells for each leukocyte population, we multiplied the percentages obtained via flow cytometry by the number of cells counted for each sample.

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

ACKNOWLEDGMENTS

We 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 Jones for providing us with his Cytopro Cytocentrifuge. Finally, we thank Leslie Tabor-Simecka and Calvin Chikelue for all the technical support and advice they provided.

FOOTNOTES

    • Received 20 April 2018.
    • Returned for modification 22 May 2018.
    • Accepted 29 June 2018.
    • Accepted manuscript posted online 9 July 2018.

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Interleukin-17A Exacerbates Disease Severity in BALB/c Mice Susceptible to Lung Infection with Mycoplasma pulmonis
Maximillion T. Mize, Xiangle L. Sun, Jerry W. Simecka
Infection and Immunity Aug 2018, 86 (9) e00292-18; DOI: 10.1128/IAI.00292-18

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Interleukin-17A Exacerbates Disease Severity in BALB/c Mice Susceptible to Lung Infection with Mycoplasma pulmonis
Maximillion T. Mize, Xiangle L. Sun, Jerry W. Simecka
Infection and Immunity Aug 2018, 86 (9) e00292-18; DOI: 10.1128/IAI.00292-18
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KEYWORDS

Th17 cells
bacterial
inflammation
cytokines
mucosa
IL-17
Mycoplasma
lung infection

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