Over 1 million people have chronic pulmonary aspergillosis secondary to tuberculosis. Furthermore, in patients with cystic fibrosis, Aspergillus fumigatus (Af) has been the most common pathogen associated with mycobacteria. The immunological basis for mycobacterial-fungal coinfection is unknown. However, mycobacterial virulence factors, like lipoarabinomannan, can disrupt host immune pathways and interfere with the clearance of intracellular Af conidia. I hypothesized that the persistence of mycobacteria in the myeloid compartment modulates the immune response against Af. Hence, I aimed to investigate the effect of the Bacillus Calmette-Guerin (BCG) and mycobacterial virulence factors on macrophage responses against Af.
Macrophages stimulated with non-mannose-capped lipoarabinomannan (LAM) showed increased cell death and inflammatory cytokine release in a dose-dependent manner. The inflammatory response was characterised by IL-1β release and by increased expression of NLRP3, Caspase-1, gasdermin-D (GSDMD) and cleaved GSDMD (GSDMD-N). Exposure to mannose-capped lipoarabinomannan (ManLAM) did not increase cell death but induced increased expression of NLRP3 and Caspase-1. Time-lapse fluorescence microscopy showed that LAM, ManLAM, and BCG decrease the capacity of macrophages to kill Af conidia within the first 6 hours of infection. Mycobacterial stimuli (LAM and ManLAM) or infection (BCG) upregulated the expression of nucleotide-binding oligomerization domain-containing protein 2 (NOD2), an intracellular sensor that activates downstream the nuclear factor kappa beta (NF-κβ). Pharmacological inhibition of NOD2 in macrophages resolved the hyperinflammatory phenotype observed in the combined infection with mycobacteria and Af.
LAM is a TLR-2 agonist, which has been shown to produce a hyperinflammatory phenotype and reduce the capacity of macrophages to clear Af. Additionally, results with BCG-Af coinfection have shown a similar hyperinflammatory response. Overall, these results suggest that mycobacteria enhance the synergic activation of TLR-2 and NOD2 in Af infection, resulting in a hyperinflammatory response. Hyperinflammation subsequently impaired the fungal killing mechanisms, activated the inflammasome, and caused pyroptotic cell death.