Clarithromycin impairs tissue-resident memory and Th17 responses to macrolide-resistant Streptococcus pneumoniae infections

Abstract

The increasing prevalence of antimicrobial resistance in pathogens is a growing public health concern, with the potential to compromise the success of infectious disease treatments in the future. Particularly, the number of infections by macrolide antibiotics-resistant Streptococcus pneumoniae is increasing. We show here that Clarithromycin impairs both the frequencies and number of interleukin (IL)-17 producing T helper (Th) 17 cells within the lungs of mice infected with a macrolide-resistant S. pneumoniae serotype 15A strain. Subsequently, the tissue-resident memory CD4⁺ T cell (Trm) response to a consecutive S. pneumoniae infection was impaired. The number of lung resident IL-17⁺ CD69⁺ Trm was diminished upon Clarithromycin treatment during reinfection. Mechanistically, Clarithromycin attenuated phosphorylation of the p90-S6-kinase as part of the ERK pathway in Th17 cells. Moreover, a strong increase in the mitochondrial-mediated maximal respiratory capacity was observed, while mitochondrial protein translation and mTOR sisgnaling were unimpaired. Therefore, treatment with macrolide antibiotics may favor the spread of antimicrobial-resistant pathogens not only by applying a selection pressure but also by decreasing the natural T cell immune response. Clinical administration of macrolide antibiotics as standard therapy procedure during initial hospitalization should be reconsidered accordingly and possibly be withheld until microbial resistance is determined. KEY MESSAGES: • Macrolide-resistant S. pneumoniae infection undergoes immunomodulation by Clarithromycin • Clarithromycin treatment hinders Th17 and tissue-resident memory responses • Macrolide antibiotics impair Th17 differentiation in vitro by ERK-pathway inhibition.

Keywords: Anti-microbial resistance; Clarithromycin; Macrolide antibiotics; Streptococcus pneumoniae; Th17 cells; Tissue-resident memory T cells.

Fig. 1

Clarithromycin treatment impairs Th17 response against S. pneumoniae infection in vivo. Mice were infected intranasally (i.n.) with 1–1.5 × 10⁶ CFU of macrolide-resistant S. pneumoniae clinical isolate (serotype 15A) under ketamine/xylazine anesthesia and treated with 10 mg/kg bodyweight Clarithromycin twice daily for 7 days. a Frequencies and b total cell numbers of IL-17A⁺ cells among CD4⁺ T cells isolated from the lung at day 7 p.i. and analyzed by FACS. c The graph shows frequencies of IFN-γ⁺ T cells. d Frequencies and e total cell number of CD69⁺ T cells stained directly after isolation from the lungs at day 7 p.i. Data shown are representative of one out of three independent experiments with 3–5 mice per group and depicted as mean ± SD. f Bacterial counts of nasal washes performed at day 2 p.i. are pooled from two independent experiments with n = 4 mice per group and depicted as geometric mean ± 95% CI. Two-tailed, unpaired Student’s t test was used to determine significance between means of groups. ns (not significantly different); *p < 0.05; **p < 0.005; ***p < 0.0005

Fig. 2

Effects of macrolide antibiotics Azithromycin and Clarithromycin on in vitro Th17 differentiation. Naïve CD4⁺ T cells were isolated from spleen and lymph nodes of mice and isolated by negative magnetic selection. Cultures under Th17 polarizing conditions were performed in 96-well plates, while 100,000 cells per well were seeded and analyzed on day 4. Graphs show a percentage of live IL-17A⁺ CD4⁺ T cells and b FoxP3⁺ T cells determined by c FACS analysis. d Cell viability is depicted as frequency of live CD4⁺ T cells. e Histogram showing CD4⁺ T cells stained with CellViolet™ to visualize proliferation at different time points. f Frequencies of divided CD4⁺ T cells upon indicated treatments at days 2 and 3 of the Th17 culture. Data shown are representative of at least three independent experiments, each performed in technical triplicates and depicted as mean ± S.E.M. Two-tailed, unpaired Student’s t test was used to determine significance between means of groups. **p < 0.005; ***p < 0.0005

Fig. 3

Analysis of mitochondrial capacity and signaling pathways of macrolide antibiotic-treated in vitro differentiated Th17 cells. a FACS analysis of mitochondrial cytochrome c oxidase depicted as mean fluorescent intensity (MFI) at day 4 of culture, data shown are representative of one out of two independent experiments, each performed in technical triplicates and depicted as mean ± S.E.M. b MFI of MitoTracker Deep Red™ at day 4 of Th17 culture, data shown are representative of one out of three independent experiments, each performed in technical duplicates and depicted as mean ± S.E.M. c Oxygen consumption rate of cells at day 3 of Th17 culture in Mito-stress test with subsequent injections of oligomycin, FCCP, rotenone, and antimycin A at the indicated time points. Data from one out of two experiments, each performed in technical triplicates and depicted as mean ± S.E.M. d Western blot of cell lysates to identify phosphorylated and unphosphorylated p70-S6-kinase at 24 h of culture, e at 45 and 60 min. Membranes were subsequently probed twice with antibodies to visualize phosphorylated and total protein as indicated with a sign plus as superscript 2. f Western blot to identify p90-S6-kinase 24 h after start of culture. g Quantification of p-p70-S6/p70-S6 and p-p90-S6/p90-S6 of three independent Western blots from independent cultures at 24 h normalized to the respective ratios in DMSO controls. All Western blots for indicated time points were run at least twice and verified initial findings. Two-tailed, unpaired Student’s t test was used to determine significance between means of groups. ns (not significantly different); *p < 0.05

Fig. 4

Clarithromycin treatment only during the first infection fails to influence Trm response to the second infection. a Graphical visualization of the experimental layout is depicted. b Frequencies of CD4⁺ T cells isolated from the lungs are shown at day 7 p.i. c Frequencies and d total cell numbers of CD4⁺CD3⁺ T cells producing IL17A upon PMA/Ionomycin restimulation are depicted. Shown are e frequencies and f total cell numbers of CD4⁺ T cells staining positive for CD69. g Representative FACS plots visualizing cytokine staining after restimulation are depicted. Data shown represent two pooled independent experiments out of three for b, c, and d (while the control group is only representative for one) and representative of one out of two for e and f with 5-3 mice per group, and depicted as mean ± S.D. Two-tailed, unpaired Student’s t test was used to determine significance between means of groups. ns (not significantly different); ***p < 0.0005

Fig. 5

Clarithromycin treatment during both infections attenuated Trm17 response. a Graphical visualization of the experimental layout is depicted. b Bodyweight curve of mice recovering from the second infection is shown as pooled data from 4 experiments. c Frequencies of CD4⁺ T cells isolated from the lungs are shown at day 7 p.i. d Frequencies and e total cell numbers of CD4⁺CD3⁺ T cells producing IL17A upon PMA/ionomycin restimulation are depicted. f Representative FACS plots visualizing cytokine staining after restimulation are depicted. Shown are g frequencies and h total cell numbers of CD69⁺CD4⁺ T. Data shown are three pooled independent experiments out of three for b–e and two pooled out of two for g and h with 3–5 mice per group and depicted as mean ± S.D. Two-tailed, unpaired Student’s t test was used to determine significance between means of groups. ns (not significantly different); *p < 0.05; **p < 0.005; ***p < 0.0005 or as indicated