Vaccination with Mycoplasma pneumoniae membrane lipoproteins induces IL-17A driven neutrophilia that mediates Vaccine-Enhanced Disease

Abstract

Bacterial lipoproteins are an often-underappreciated class of microbe-associated molecular patterns with potent immunomodulatory activity. We previously reported that vaccination of BALB/c mice with Mycoplasma pneumoniae (Mp) lipid-associated membrane proteins (LAMPs) resulted in lipoprotein-dependent vaccine enhanced disease after challenge with virulent Mp, though the immune responses underpinning this phenomenon remain poorly understood. Herein, we report that lipoprotein-induced VED in a mouse model is associated with elevated inflammatory cytokines TNF-α, IL-1β, IL-6, IL-17A, and KC in lung lavage fluid and with suppurative pneumonia marked by exuberant neutrophilia in the pulmonary parenchyma. Whole-lung-digest flow cytometry and RNAScope analysis identified multiple cellular sources for IL-17A, and the numbers of IL-17A producing cells were increased in LAMPs-vaccinated/Mp-challenged animals compared to controls. Specific IL-17A or neutrophil depletion reduced disease severity in our VED model-indicating that Mp lipoproteins induce VED in an IL-17A-dependent manner and through exuberant neutrophil recruitment. IL-17A neutralization reduced levels of TNF-α, IL-1β, IL-6, and KC, indicating that IL-17A preceded other inflammatory cytokines. Surprisingly, we found that IL-17A neutralization impaired bacterial clearance, while neutrophil depletion improved it-indicating that, while IL-17A appears to confer both maladaptive and protective responses, neutrophils play an entirely maladaptive role in VED. Given that lipoproteins are found in virtually all bacteria, the potential for lipoprotein-mediated maladaptive inflammatory responses should be taken into consideration when developing vaccines against bacterial pathogens.

Fig. 1. Vaccination with M. pneumoniae lipoproteins is associated with potent reactivation of inflammatory cytokines responses after challenge.

A Illustration of experimental timeline and outcome measures. B Lung lesion scores of vaccinated-then-challenged animals. Bronchoalveolar lavage fluid (BALF) concentrations of C TNF-α, D IL-1β, E IL-6, F IL-17A, and G KC in vaccinated-then-challenged animals. Positive correlations between disease severity (lung lesion scores) and BALF H IL-17A, and I KC concentrations. *p < 0.5, **p < 0.1, ***p < 0.01, ****p < 0.001. Error bars for B indicate median and interquartile range and mean and SEM for C–G. Dotted lines for linear regression graphs indicate 95% confidence intervals. Each point represents data from an individual animal. Nonparametric lesion score data were analyzed via a one-way ANOVA on ranks (Kruskal–Wallis) with a Dunn’s post-hoc test for multiple pairwise comparisons. Parametric cytokine concentration data were analyzed via an ordinary one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise comparisons. Linear regression was utilized to establish correlations.

Fig. 2. Numbers of IL-17A producing cells are elevated in LAMPs-vaccinated/Mp-challenged animals.

Percentage (A, E, I) and number (B, F, J) of IL-17A positive cells when analyzing all live-single-cells (top), lymphocyte-like live-single cells (middle) and other-cells (bottom). Overlaid histograms (C, G, K) of cell count vs IL-17A signal on all live-single-cells (top), lymphocyte-like live-single cells (middle) and other-cells (bottom). Dot-plots (D, H, L) of Forward Scatter area vs IL-17A signal on all live-single-cells (top), lymphocyte-like live-single cells (middle) and other-cells (bottom). *p < 0.5, **p < 0.1, ***p < 0.01, ****p < 0.001. Error bars indicate mean and SEM. Each point represents data from an individual animal. Data from single representative animals from each vaccination group are shown on histograms and dot-plots. Nonparametric percent frequency data were analyzed via a one-way ANOVA on ranks (Kruskal–Wallis) with a Dunn’s post-hoc test for multiple pairwise comparisons. Parametric cell count data were analyzed via an ordinary one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise comparisons.

Fig. 3. Numbers and percentages of IL-17A producing lymphocyte-like cells based on expression of CD3 and CD4.

IL-17A producing CD3 + CD4 + cells as a percent of IL-17A positive lymphocyte-like cells (A) and raw counts per 50k analyzed events (E). IL-17A producing CD3 + CD4- cells as a percent of IL-17A positive lymphocyte-like cells (B) and raw counts per 50k analyzed events (F). IL-17A producing CD3-CD4 + cells as a percent of IL-17A positive lymphocyte-like cells (C) and raw counts per 50k analyzed events (G). IL-17A producing CD3-CD4- cells as a percent of IL-17A positive lymphocyte-like cells (D) and raw counts per 50k analyzed events (E). *p < 0.5, **p < 0.1, ***p < 0.01, ****p < 0.001. Error bars indicate mean and SEM. Each point represents data from an individual animal. Nonparametric percent frequency data were analyzed via a one-way ANOVA on ranks (Kruskal–Wallis) with a Dunn’s post-hoc test for multiple pairwise comparisons. Parametric cell count data were analyzed via an ordinary one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise comparisons.

Fig. 4. H&E and RNAScope in-situ hybridization lung histology from vaccinated-then-challenged animals.

Representative H&E stained lung sections (A, C, E, G, I, K) and RNAScope in situ hybridization processed slides staining IL-17A transcript (blue) and CD4 transcript (red) (B, D, F, H, J, L) from Sham-vaccinated/Mp-challenged animals (top), LAMPs-vaccinated/Mp-challenged animals (middle) and dLAMPs-vaccinated/Mp-challenged animals (bottom). Scale bars indicate 500 um (4x) or 100 um (10x).

Fig. 5. H&E and RNAScope in situ hybridization lung histology highlighting peribronchiolar and perivascular lesions.

H&E stained lung sections (left) and RNAScope in situ hybridization processed slides staining IL-17A transcript (blue) and CD4 transcript (red) (right) displaying vessels and airways to show that CD4 mRNA and IL-17A mRNA co-localization was more frequent in the areas of perivascular cuffing.

Fig. 6. Vaccination with M. pneumoniae lipoproteins is associated with enhanced lung neutrophilia upon challenge, and this neutrophilia is associated with more severe disease.

A Illustration of experimental timeline and outcome measures. B Numbers of lung-infiltrating leukocytes, proportion C and numbers D of lung-infiltrating neutrophils in vaccinated-then-challenged animals. E Positive correlations between lung-infiltrating neutrophil proportions and Lung Lesion Scores. F–I Correlations between proportions and numbers of lung-infiltrating neutrophils and IL-17A and KC concentrations. *p < 0.5, **p < 0.1, ***p < 0.01, ****p < 0.001. Error bars for B–D indicate mean and SEM. Dotted lines for linear regression graphs indicate 95% confidence intervals. Each point represents data from an individual animal. Nonparametric percent frequency/proportion data were analyzed via a one-way ANOVA on ranks (Kruskal–Wallis) with a Dunn’s post-hoc test for multiple pairwise comparisons. Parametric cell count data were analyzed via an ordinary one-way ANOVA with a Tukey’s post-hoc test for multiple pairwise comparisons. Linear regression was utilized to establish correlations.

Fig. 7. IL-17A neutralization in LAMPs-vaccinated/Mp-challenged animals reduces inflammatory cytokines, neutrophil recruitment, and severity of histopathological lung lesions but impairs bacterial clearance.

A Illustration of experimental timeline and outcome measures. BALF concentrations of B IL-17A, C TNF-α, D IL-1β, E IL-6, and F KC in LAMPs-vaccinated/Mp-challenged animals receiving an anti-IL-17A neutralizing monoclonal antibody (17F3) or isotype control (MOPC-21). BALF numbers of lung-infiltrating leukocytes (G), and proportion of H and number of (I) lung-infiltrating neutrophils. J Lung Lesion Scores and K bacterial loads of vaccinated-then-challenged animals treated with anti-IL-17A antibody or isotype control. *p < 0.5, **p < 0.1, ***p < 0.01, ****p < 0.001. Error bars for J and K indicate median and interquartile range and mean and SEM for B–I. Each point represents data from an individual animal. Nonparametric lesion score, bacterial burden and percent frequency/proportion data were analyzed via an unpaired, two-tailed Mann–Whitney U-test. Parametric cytokine concentration and cell count data were analyzed via an unpaired, two-tailed t-test.

Fig. 8. Neutrophil depletion in LAMPs-vaccinated/Mp-challenged mice ameliorates disease severity and enhances bacterial clearance.

A Illustration of experimental timeline and outcome measures. Anti-Ly6G (1A8) antibody is given as a daily injection and its isotype control clone 2A3 is given to control animals following the same schedule. To induce an isotype switch of the 1A8 antibody for sustained neutrophil depletion, the anti-rat K (MAR18.5) antibody was given every-other day, while the isotype control MOPC-21 was given at the same schedule to control animals. To achieve sustained depletion of neutrophils BALF numbers of lung-infiltrating leukocytes (B), and proportion of C and number of D lung-infiltrating neutrophils in LAMPs-vaccinated/Mp-challenged animals receiving either neutrophil depletion antibodies (1A8-anti-Ly6G) or isotype control (2A3). BALF concentrations of F TNF-α and G KC in LAMPs-vaccinated/Mp-challenged animals receiving neutrophil depleting antibodies or isotype control. E Lung Lesion Scores and H bacterial loads of vaccinated-then-challenged animals treated with neutrophil depleting antibody or isotype control. *p < 0.5, **p < 0.1, ***p < 0.01, ****p < 0.001. Error bars for E and H indicate median and interquartile range and mean and SEM for B–D and F, G. Each point represents data from an individual animal. Nonparametric lesion score, bacterial burden and percent frequency/proportion data were analyzed via an unpaired, two-tailed Mann–Whitney U-test. Parametric cytokine concentration and cell count data were analyzed via an unpaired, two-tailed t-test.

Fig. 9. Putative model of M. pneumoniae Vaccine-Enhanced Disease.

Anamnestic reactivation of IL-17A recall responses in LAMPs-vaccinated/Mp-challenged animals results in the further production of TNF-α, IL-1β, IL-6, and KC. TNF-α and IL-1β can further induce the expression of the neutrophil chemotactic factor KC (Supplementary References 1,2), and in the presence of IL-6, further potentiate IL-17A production by helper T-cells (Supplementary Reference 3), establishing a positive feedback loop of neutrophil recruitment and inflammation. Neutrophils also contribute to TNF-α production which can further potentiate KC production, contributing to the positive neutrophil recruitment loop that is associated with the more severe disease observed in Mp VED. (Created in biorender.com by ABM).