The C3-C3aR axis modulates trained immunity in alveolar macrophages

Reviewer #1 (Public review):

Summary:

This study is built on the emerging knowledge of trained immunity, where innate immune cells exhibit enhanced inflammatory responses upon being challenged by a prior insult. Trained immunity is now a very fast-evolving field and has been explored in diverse disease conditions and immune cell types. Earhart and the team approached the topic from a novel angle and were the first to explore a potential link to the complement system.

The study focused on the central complement protein C3 and investigated how its signalling may modulate immune training in alveolar macrophages. The authors first performed in vivo experiments in C57BL mouse models to observe the presence of enhanced inflammation and C3a in BAL fluid following immune training. These changes were then compared with those from C3-deficient mice, which confirmed the involvement of C3a. This trained immunity was further validated in ex vivo experiments using primary alveolar macrophage, which was blunted in C3-deficiency, and, intriguingly, rescued by adding exogenous C3 protein, but not C3a. The genetic-based findings were supported by pharmacological experiments using the C3aR antagonist SB290157. Mechanistically, transcriptomic analyses suggested the involvement of metabolism-linked, particularly glycolytic, genes, which was in agreement with an upregulation of glycolytic flux in WT but not C3-deficient macrophages.

Collectively, these data suggest that C3, possibly through engaging with C3aR, contributes to trained immunity in alveolar macrophages.

Strengths:

The conclusions reached were well supported by in vivo and ex vivo experiments, encompassing both genetic-knockout animal models and pharmacological tools.

The transcriptomic and cell metabolism studies provided valuable mechanistic insights.

Weaknesses:

For the in vivo experiments, the histopathological and other inflammatory markers (Figure 1) were not directly linked to alveolar macrophages by experimental evidence. Other innate immune cells (eg. dendritic cells, neutrophils) and endothelial cells could also be involved in immune training and contribute to the pathological outcomes. These cells were not examined or mentioned in the study.

For the ex vivo experiments assessing immune training in alveolar macrophages, only the release of selected inflammatory factors were measured. Macrophage activities constitute multiple aspects (e.g. phagocytosis, ROS production, microbe killing), which should also be considered to better depict the effect of trained immunity.

The proposed mechanism of C3 getting cleaved intracellularly and then binding to lysosomal C3aR needs to be further supported by experimental evidence.

There was an absence of any validation in human-based models.

Reviewer #2 (Public review):

Earhart et al. investigated the role of the complement system in trained innate immunity (TII) in alveolar macrophages (AM). They used a WT and C3 knockout murine model primed with locally administered heat-killed P. aeruginosa (HKPA). Additionally, they employed ex vivo AM training models using C3 knockout mice, where reconstitution of C3 and blockade of C3R were performed. The study concluded that the C3-C3R axis is essential for inducing TII in macrophages in the ex vivo model. The manuscript is well-written and easy to follow. However, I have the following major concerns.

(1) The secondary challenge to assess the reprogramming of innate cells in the BAL was conducted 14 days after the initial exposure to HKPA. However, no evidence is provided to confirm that homeostasis was re-established following the primary exposure. Demonstrating the resolution of acute inflammation is essential to ensure that the observed responses to the secondary challenge are not confounded by persistent inflammation from the initial exposure.

(2) In Figure 1D, cytokine production by BAL cells from WT and C3KO mice after HKPA exposure and LPS challenge is shown. However, it is unclear whether the reduced response in trained C3KO mice is due to a defect in trained immunity or an intrinsic inability of C3KO cells to respond to LPS. To clarify this, the response of trained C3KO cells should also be compared to untrained C3KO controls after the LPS challenge. This comparison is necessary to determine if the reduction is specifically related to innate immune memory or a broader impairment in LPS responsiveness. Such control should be included in all ex vivo training and LPS stimulation experiments as well.

(3) The data presented provide evidence of alterations in the functional and metabolic activities of innate cells in the lung, indicating the induction of innate immune memory in a C3-C3R axis-dependent pathway. However, it remains to be established whether such changes can lead to altered disease outcomes. Therefore, the impact of these changes should be demonstrated, for instance, through an infection model to support the claim made in the study that C3 modulates trained immunity in AMs through C3aR signalling.

(4) Figure 3, panels B and C - stats should be shown for comparing WT-HKPA-trained and C3KO HKPA-trained.

(5) In Figure 4, where the proper untrained C3KO is included, the data presented in Figure 4C show an increase in basal and maximum glycolysis in trained C3KO compared to their untrained control counterparts. Statistical analysis should be provided for this comparison. Based on these data, it appears that metabolic reprogramming occurs even in the absence of C3. Furthermore, C3KO cells intrinsically exhibit reduced glycolytic capacity compared to WT. These observations challenge the conclusions made in the manuscript. Therefore, without the proper control (untrained C3KO) included in all experimental approaches, it is impossible to draw an evidence-based conclusion that the C3-C3R axis plays a role in the induction of innate immune memory.

(6) The Results and Discussion sections should be separated, and the results should be thoroughly analyzed in the context of published literature. Separating these sections will allow for a clearer presentation of findings and ensure that the discussion provides a comprehensive interpretation of the data.

Author response:

We thank both reviewers for their suggestions on improving our manuscript, which is focused on demonstrating that the C3a-C3aR axis modulates trained immune responses in alveolar macrophages. The Short Report format precludes separating the Results and Discussion sections. However, we will work towards a clearer presentation of findings and providing a more comprehensive interpretation of the data in the Revision, by addressing the points brought up by both Reviewers.

We agree with the suggestions from Reviewer 1 that (1) other cell types such as dendritic cells, neutrophils, and endothelial cells can also be involved in immune training, and (2) macrophages have other activities beyond releasing inflammatory cytokines, and will clarify both these points in the Revision. The mechanism of C3 being cleaved intracellularly and binding to lysosomal C3aR involves cathepsin-dependent cleavage of C3 to C3a and has been experimentally proven (Liszewski et al. Immunity 2013). However, we will clarify this mechanism in the revision. We also acknowledge that the observations need to be validated in human-based models. Currently, we do not have access to an adequate representation of human alveolar macrophages for our ex vivo testing to account for individual-level variation in immune responses. However, we anticipate this work will form the basis of these future studies.

We also appreciate Reviewer 2’s suggestions regarding demonstrating the resolution of acute inflammation after the initial exposure to heat-killed Pseudomonas. We will address this critique by performing additional experiments, which will be included in the Revision. We also agree that the responses of trained C3-deficient cells should be compared to untrained C3-deficient controls after the LPS challenge. We will include this data in the Revision, in addition to the requested data for Figures 3 and 4. We would like to clarify that we do not observe baseline differences between untrained C3-sufficient (wildtype) and C3-deficient alveolar macrophages, even in their glycolytic capacity, and thus, anticipate that our revised data will strengthen the conclusions from the original manuscript.