Human airway macrophages are metabolically reprogrammed by IFN-γ resulting in glycolysis dependent functional plasticity

Reviewer #1 (Public Review):

Summary:

The researchers demonstrated that when cytokine priming is combined with exposure to pathogens or pathogen-associated molecular patterns, human alveolar macrophages and monocyte-derived macrophages undergo metabolic adaptations, becoming more glycolytic while reducing oxidative phosphorylation. This metabolic plasticity is greater in monocyte-derived macrophages than in alveolar macrophages.

Strengths:

This study presents evidence of metabolic reprogramming in human macrophages, which significantly contributes to our existing understanding of this field primarily derived from murine models.

Weaknesses:

The study has limited conceptual novelty.

Reviewer #2 (Public Review):

Summary:

The authors aimed to functionally characterize primary human airway macrophages and monocyte-derived macrophages, correlating their glycolytic shift in metabolism. They conducted this macrophage characterization in response to type II interferon and IL-4 priming signals, followed by different stimuli of irradiated Mycobacterium tuberculosis and LPS.

Strengths:

(1) The study employs a thorough measurement of metabolic shift in metabolism by assessing extracellular acidification rate (ECAR) and oxygen consumption rate (OCR) of differentially polarized primary human macrophages using the Seahorse XFe24 Analyzer.
(2) The effect of differential metabolic shift on the expression of different surface markers for macrophage activation is evaluated through immunofluorescence flow cytometry and cytokine measurement via ELISA.
(3) The authors have achieved their aim of preliminarily characterizing the glycolysis-dependent cytokine profile and activation marker expression of IFN-g and IL-4 primed primary human macrophages.
(4) The results of the study support its conclusion of glycolysis-dependent phenotypical differences in cytokine secretion and activation marker expression of AMs and MDMs.

Weaknesses:

(1) The data are presented in duplicates for cross-analyses.
(2) The data presented supports a distinct functional profile of airway macrophages (AMs) compared to monocyte (blood)-derived macrophages (MDMs) in response to the same priming signals. However, the study does not attempt to explore the underlying mechanism for this difference.
(3) The study is descriptive in nature, and the results validate IFN-g-mediated glycolytic reprogramming in primary human macrophages without providing mechanistic insights.

Reviewer #3 (Public Review):

Summary:

In this manuscript, the authors explore the contribution of metabolism to the response of two subpopulations of macrophages to bacterial pathogens commonly encountered in the human lung, as well as the influence of priming signals typically produced at a site of inflammation. The two subpopulations are resident airway macrophages (AM) isolated via bronchoalveolar lavage and monocyte-derived macrophages (MDM) isolated from human blood and differentiated using human serum. The two cell types were primed using IFNγ and Il-4, which are produced at sites of inflammation as part of initiation and resolution of inflammation respectively, followed by stimulation with either irradiated Mycobacterium tuberculosis (Mtb) or LPS to simulate interaction with a bacterial pathogen. The authors use human cells for this work, which makes use of widely reported and thoroughly described priming signals, as well as model antigens. This makes the observations on the functional response of these two subpopulations relevant to human health and disease. To examine the relationship between metabolism and functional response, the authors measure rates of oxidative phosphorylation and glycolysis under baseline conditions, primed using IFNγ or IL-4, and primed and stimulated with Mtb or LPS.

Strengths:

• The data indicate that both populations of macrophages increase metabolic rates when primed, but MDMs decrease their rates of oxidative phosphorylation after IL-4 priming and bacterial exposure while AMs do not.
• It is demonstrated that glycolysis rates are directly linked to the expression of surface molecules involved in T-cell stimulation and while secretion of TNFα in AM is dependent on glycolysis, in MDM this is not the case. IL-1β is regulated by glycolysis only after IFN-γ priming in both MDM and AM populations. It is also demonstrated that Mtb and LPS stimulation produces responses that are not metabolically consistent across the two macrophage populations. The Mtb-induced response in MDMs differed from the LPS response, in that it relies on glycolysis, while this relationship is reversed in AMs. The difference in metabolic contributions to functional outcomes between these two macrophage populations is significant, despite acknowledgement of the reductive nature of the system by the authors.
• The observations that AM and MDM rely on glycolysis for the production of cytokines during a response to bacterial pathogens in the lung, but that only MDM shift to Warburg Metabolism, though this shift is blocked following exposure to IL-4, are supported by the data and a significant contribution the study of the innate immune response.

Weaknesses:

• It is unclear whether changes in glycolysis and oxidative phosphorylation in primed cells are due to priming or subsequent treatments. ECAR and OCR analyses were therefore difficult to interpret.
• The data may not support a claim that AM has greater "functional plasticity" without a direct comparison of antigen presentation. Moreover, MDM secrete more IL-1β than AM. The claim that AM "have increased ability to produce all cytokines assayed in response to Mtb stimulation" does not appear to be supported by the data.
• The claim that AM are better for "innate training" via IFNγ may not be consistent with increased IL-1β and a later claim that MDM have increased production and are "associated with optimal training."
• Statistical analyses may not appropriately support some of the conclusions.
• AM populations would benefit from further definition-presumably this is a heterogenous, mixed population.
• The term "functional plasticity" could also be more stringently defined for the purposes of this study.

Conclusion:

Overall, the authors succeed in their goals of investigating how inflammatory and anti-inflammatory cytokine priming contributes to the metabolic reprogramming of AM and MDM populations. Their conclusions regarding the relationship between cytokine secretion and inflammatory molecule expression in response to bacterial stimuli are supported by the data. The involvement of metabolism in innate immune cell function is relevant when devising treatment strategies that target the innate immune response during infection. The data presented in this paper further our understanding of that relationship and advance the field of innate immune cell biology.