Cyclooxygenase inhibitors impair CD4 T cell immunity and exacerbate Mycobacterium tuberculosis infection in aerosol-challenged mice

Tuberculosis, caused by infection with Mycobacterium tuberculosis (Mtb), kills over 1.6 million people each year despite availability of antibiotics. The increase in drug resistant Mtb strains is a major public health emergency and host-directed therapy as adjunct to antibiotic treatment has gained increased interest. Cyclooxygenase inhibitors (COXi) are frequently used drugs to alleviate tuberculosis related symptoms. Mouse studies of acute intravenous Mtb infection have suggested a potential benefit of COXi for host-directed therapy. Here we show that COXi treatment (ibuprofen and celecoxib) is detrimental to Mtb control in different mouse models of respiratory infection. This effect links to impairments of the Type-1 helper (Th1) T-cell response as CD4 T-cells in COXi-treated animals have significantly decreased Th1 differentiation, reduced IFNγ expression and decreased protective capacity upon adoptive transfer. If confirmed in clinical trials, these findings could have major impact on global health and question the use of COXi for host-directed therapy.


Statistics
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Life sciences study design
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Sample size
In the standardized murine aerosol challenge model (incl.the adoptive transfer study), we powered the experiments for detecting a treatment effect 0.5 log10 CFU reduction in lungs compared to non-treated controls with a type I error rate of 5% (α=0.05), a power of 80% and standard deviation of 0.35 log10 CFU (based on previous experiments).This results in n=8 mice per group for primary analysis.We had no prior experience with the intravenous infection model and no power calculations were made.To ensure conclusive results, we increased the samplesize to n=13.The same goes for the high-dose challenge study where n=32 was selected due to the binary endpoint of "dead/alive".In the re-infection model, we powered the experiment for detecting a treatment effect of 0.5 log10 CFU reduction compared to non-treated controls with a type I error rate of 5% (α=0.05), a power of 80% and standard deviation of 0.5 log10 CFU.This results in n=16 mice per group for primary analysis.
Data exclusions No data points was excluded from the analysis

Replication
The main finding of this paper, is that COXi treatment increase the bacterial burden after Mtb aerosol challenge in mice.We demonstrate this effect in Five different experimental setups (a total of six experiments): 1. celecoxib treatment in the stadardized aerosol infection model (one experiment), 2. ibuprofen (different COXi) treatment in the stardized aerosol infection model (two experiments),3 .Ibuprofen treatment during lethal high-dose aerosol infection 4. celecoxib treatment in a re-infection model (one experiment) and 5. adoptive transfer of CD4 T cells from celecoxib treated mice (one experiment).Our appraoch of using five different experimental set-ups (reflecting four different aspects of the treatment effect) ensures robustness of our findings.Since our results contradicted previous published studies, we validated that we could replicate their data when we adapted their experimental approach (two experiments with intravenous infection).
Randomization Mice were randomly assigned to cages upon arrival to our animal facility.For each time point in the study, mice were randomly selected for primary analysis.

Blinding
The investigator was not involved in data collection.Organ homogenization, plating and CFU counting was performed by an experienced technician, who was not involved in study design and/or data analysis.Changes in histopathology was performed by a skilled pathologist who was blinded for both group association and treatment.The investigator was not blinded during data analysis and interpretation.
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Validation
Validation in the target species was performed by the supplier of the antibody.