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.

Sometimes the sentences are very long and difficult to read; I suggest improving the text, following this advice (lines 87-91: sentence too long, furthermore, there is "evaluated" twice; lines 129-134.

Results
Lines 138-167: In order to more easily understand the results, I think that in the manuscript the same indication regarding the days of treatment (of infection) in which the BALF was performed should be indicated both in the text and in the Figures. For example, in line 144 "day 15, 24 and 36 from infection" correspond to 8, 17, 29 days of treatment. I think that it could generate confusion in the reader. Interesting results were obtained but maybe in this way they could be more understandable.
Lines 169-197: The treatment is explained well in Material and methods, but I think it is important to remind the conditions at the beginning of this paragraph. The treatment includes a combination of liposomes (intra-nasal administration) and amikacin (ip administration).

Minor revisions
Line 41: BALF: bronchoalveolar lavage fluid. Please write in extenso and put the abbreviation in brackets. Lines 107-109: explain better the differences among the results showed in Fig. 2S and Fig. 1A. Lines 123-124: you have already introduced the abbreviation CF; so, you can write "CF patients". Lines 130-131: responsible for... Line 246: proinflammatory Line 277: these? Line 320: were instead of was. Line 413: -80{degree sign}C Please change "u" with " " in ug/ml. General comment The manuscript of Poerio N. et al is interesting. The Authors report data on ex vivo and in vivo activity of a novel therapeutic regimen based on bioactive liposomes plus amikacin against Mycobacterium abscessus infection. Experimental design is appropriate, and results clearly presented. Page 28, line 556: explain the effect of Concanamycin A on phagolysosome acidification. Page 30, line 589: "amikacin", instead of "AMK". I also suggest using "amikacin" instead of "Amikacin" throughout the manuscript, wherever appropriate. Supplementary Table 1. Indicate the meaning of FEV-1, and explain while the age of each patient was indicated as age range.
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The increase of nontuberculous mycobacteria infections and the lack of an adequate antibiotic therapy represent a major health problem. This manuscript represents an innovative approach to fight this pathogen. The authors validated their approach in vivo also in a CF murine model; the results of the study are supported by experimental data. The manuscript is well written and technically sound. I would like to recommend it for the publication after minor revisions and suggestions.
Please, check the English language style through the full manuscript because there are some typos (e.g.: singular/plural, etc).
Check the italics (species, in vivo, in vitro, etc). Furthermore, the first time that one species is introduced, has to be written also in extenso (e.g.: Mycobacterium tuberculosis).
Sometimes the sentences are very long and difficult to read; I suggest improving the text, following this advice (lines 87-91: sentence too long, furthermore, there is "evaluated" twice; lines 129-134.

Results
Lines 138-167: In order to more easily understand the results, I think that in the manuscript the same indication regarding the days of treatment (of infection) in which the BALF was performed should be indicated both in the text and in the Figures. For example, in line 144 "day 15, 24 and 36 from infection" correspond to 8, 17, 29 days of treatment. I think that it could generate confusion in the reader. Interesting results were obtained but maybe in this way they could be more understandable.
Lines 169-197: The treatment is explained well in Material and methods, but I think it is important to remind the conditions at the beginning of this paragraph. The treatment includes a combination of liposomes (intranasal administration) and amikacin (ip administration).

Minor revisions
Line 41: BALF: bronchoalveolar lavage fluid. Please write in extenso and put the abbreviation in brackets. Lines 107-109: explain better the differences among the results showed in Fig. 2S and Fig. 1A. Lines 123-124: you have already introduced the abbreviation CF; so, you can write "CF patients". Lines 130-131: responsible for… Line 246: proinflammatory Line 277: these? Line 320: were instead of was. Line 413: -80°C Please change "u" with "" in ug/ml.