Combined Host- and Pathogen-Directed Therapy for the Control of Mycobacterium abscessus Infection

Abstract

Mycobacterium abscessus is the etiological agent of severe pulmonary infections in vulnerable patients, such as those with cystic fibrosis (CF), where it represents a relevant cause of morbidity and mortality. Treatment of pulmonary infections caused by M. abscessus remains extremely difficult, as this species is resistant to most classes of antibiotics, including macrolides, aminoglycosides, rifamycins, tetracyclines, and β-lactams. Here, we show that apoptotic body like liposomes loaded with phosphatidylinositol 5-phosphate (ABL/PI5P) enhance the antimycobacterial response, both in macrophages from healthy donors exposed to pharmacological inhibition of cystic fibrosis transmembrane conductance regulator (CFTR) and in macrophages from CF patients, by enhancing phagosome acidification and reactive oxygen species (ROS) production. The treatment with liposomes of wild-type as well as CF mice, intratracheally infected with M. abscessus, resulted in about a 2-log reduction of pulmonary mycobacterial burden and a significant reduction of macrophages and neutrophils in bronchoalveolar lavage fluid (BALF). Finally, the combination treatment with ABL/PI5P and amikacin, to specifically target intracellular and extracellular bacilli, resulted in a further significant reduction of both pulmonary mycobacterial burden and inflammatory response in comparison with the single treatments. These results offer the conceptual basis for a novel therapeutic regimen based on antibiotic and bioactive liposomes, used as a combined host- and pathogen-directed therapeutic strategy, aimed at the control of M. abscessus infection, and of related immunopathogenic responses, for which therapeutic options are still limited. IMPORTANCE Mycobacterium abscessus is an opportunistic pathogen intrinsically resistant to many antibiotics, frequently linked to chronic pulmonary infections, and representing a relevant cause of morbidity and mortality, especially in immunocompromised patients, such as those affected by cystic fibrosis. M. abscessus-caused pulmonary infection treatment is extremely difficult due to its high toxicity and long-lasting regimen with life-impairing side effects and the scarce availability of new antibiotics approved for human use. In this context, there is an urgent need for the development of an alternative therapeutic strategy that aims at improving the current management of patients affected by chronic M. abscessus infections. Our data support the therapeutic value of a combined host- and pathogen-directed therapy as a promising approach, as an alternative to single treatments, to simultaneously target intracellular and extracellular pathogens and improve the clinical management of patients infected with multidrug-resistant pathogens such as M. abscessus.

Keywords: chronic infection; cystic fibrosis; host-pathogen interactions; infectious disease; innate immunity; liposomes; nontuberculous mycobacteria; pulmonary infection.

FIG 1

ABL/PA, ABL/PI3P, and ABL/PI5P enhance both internalization and intracellular killing of M. abscessus in dTHP-1 cells and monocyte-derived macrophages (MDM) from healthy donors (HD) with pharmacologically inhibited CFTR. (A to C) dTHP-1 cells (A and B) and primary MDM from healthy donors (C), treated or not treated with INH172, were cultured at 5 × 10⁵ cells/well in 24-well plates and 2 × 10⁵ cells/well in 96-well plates. (A) Cells were stimulated with selected ABL formulations before infection for 30 min and then infected with the M. abscessus reference strain (ATCC 19977). Bacterial uptake was quantified by CFU assay and indicated as phagocytosis index, calculated as the ratio between the CFU obtained immediately after the infection and those from the inoculum. (B and C) Cells, dTHP-1 cells (B) and MDM from healthy donors (C), were exposed or not exposed to INH172, infected with M. Abscessus, and treated for 18 h with the selected ABL formulations. Bacterial growth was assessed by CFU assay. The replication index was calculated as the ratio between the CFU obtained 18 h after infection, in the presence or absence of ABL formulations, and the CFU obtained before the addition of liposomes. The results are shown as the mean ± standard deviation of the values obtained from triplicates of each condition, and panel C is representative of experiments with cells from four different donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001 by Student’s t test.

FIG 2

ABL/PI5P promotes both ROS- and phagolysosome acidification-dependent intracellular M. abscessus killing in CF MDM. (A to C) MDM isolated from CF patients (n = 17) were plated at the concentration of 1 × 10⁶ cells/mL, infected with the M. abscessus reference strain (ATCC 19977), and then stimulated for 18 h with ABL carrying PI5P (A) (n = 17) in the presence or absence of catalase (PEG-Cat) (B) (representative of n = 4) or concanamycin A (Conc A), a specific inhibitor of vacuolar type H⁺-ATPase activity (C) (representative of n = 4) at the concentration of 100 U/mL or 1 nM, respectively. Bacterial growth was assessed by CFU assay, and the replication index was calculated as the ratio between the CFU obtained 18 h after infection, in the presence or absence of ABL formulations, and the CFU obtained before the addition of liposomes. (A) Statistical analysis was performed using the two-sided Wilcoxon matched-pair signed rank test (P = 0.0005). (B and C) The results are shown as the mean ± standard deviation of the values obtained from each condition and are representative of experiments with cells from four different CF patients. *, P < 0.05; **, P < 0.01, ***, P < 0.001; ****, P < 0.0001 by Student’s t test.

FIG 3

ABL/PI5P reduces both bacterial burden and leukocyte recruitment in WT mice infected with M. abscessus. (A) C57BL/6N mice were chronically infected with 10⁵ CFU of the M. abscessus reference strain (ATCC 19977) by intratracheal (i.t.) injection and treated after 1 week of infection with ABL loaded with PA, PI3P, or PI5P three times a week for 8, 17, and 29 days. (A and B) After these time points mice were sacrificed, and BALF and lungs were processed for microbiological analysis (B) and evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment (C). Data from two independent experiments were pooled. The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01).

FIG 4

ABL/PI5P reduces both bacterial burden and leukocyte recruitment in CF mice infected with M. abscessus. (A) WT and CF mice were chronically infected with 10⁵ CFU of the M. abscessus reference strain (ATCC 19977) by i.t. injection and treated with ABL/PI5P three times a week for 8 and 29 days. (B and C) Mice were then sacrificed, and BALF and lungs were processed for (B) microbiological analysis and (C) evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment to establish the therapeutic efficacy of liposomes. The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01).

FIG 5

ABL/PI5P-amikacin combined treatment promotes higher reduction of M. abscessus intracellular growth than single treatments in CF MDM. CF MDM were cultured and infected as described in Materials and Methods. (A and B) Finally, supernatant was collected, cells were lysed, and both were analyzed for extracellular (A) and intracellular (B) bacterial growth. The replication index was calculated as the ratio between the CFU obtained 18 h after infection in the presence or absence of ABL/PI5P and/or amikacin (AMK) and those obtained immediately after infection, before the addition of the stimuli. Results are shown as the mean ± standard deviation of the values obtained from triplicates of each condition and are representative of four CF patients. n.s., not significant; *, P < 0.05; ***, P < 0.001; ****, P < 0.0001 by Student’s t test.

FIG 6

ABL/PI5P-amikacin combined treatment induces both M. abscessus clearance and anti-inflammatory responses in WT mice. (A) WT mice were chronically infected with 10⁵ CFU of the M. abscessus reference strain (ATCC 19977) and treated 1 week after infection with AMK and/or ABL/PI5P as described in Materials and Methods. (B and C) After 8 and 29 days of treatment, mice were sacrificed, and BALF and lungs were processed for microbiological analysis (B) and evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment (C). The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).

FIG 7

ABL/PI5P-amikacin combined treatment induces both M. abscessus clearance and anti-inflammatory responses in CF mice. (A) CF mice were chronically infected with 10⁵ CFU of the M. abscessus reference strain (ATCC 19977) and treated 1 week after infection with AMK and/or ABL/PI5P as described in Materials and Methods. (B and C) After 29 days of treatment mice were sacrificed, and BALF and lungs were processed for microbiological analysis (B) and evaluation of neutrophil, macrophage, and lymphocyte BALF recruitment (C). The results are shown as the median of the values, and statistical significance by Mann-Whitney test was determined (*, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001).