RAS-p110a signalling in macrophages is required for effective inflammatory response and resolution of inflammation

Reviewer #1 (Public review):

This study by Alejandro Rosell et al. reveals the immunoregulatory role of the RAS-p110α pathway in macrophages, specifically in regulating monocyte extravasation and lysosomal digestion during inflammation. Disrupting this pathway, through genetic tools or pharmacological intervention in mice, impairs the inflammatory response, leading to delayed resolution and more severe acute inflammation. The authors suggest that activating p110α with small molecules could be a potential therapeutic strategy for treating chronic inflammation. These findings provide important insights into the mechanisms by which p110α regulates macrophage function and the overall inflammatory response.

The updates made by the authors in the revised version have addressed the main points raised in the initial review, further improving the strength of their findings.

Reviewer #2 (Public review):

Summary:

Cell intrinsic signaling pathways controlling the function of macrophages in inflammatory processes, including in response to infection, injury or in the resolution of inflammation are incompletely understood. In this study, Rosell et al. investigate the contribution of RAS-p110α signaling to macrophage activity. p110α is a ubiquitously expressed catalytic subunit of PI3K with previously described roles in multiple biological processes including in epithelial cell growth and survival, and carcinogenesis. While previous studies have already suggested a role for RAS-p110α signaling in macrophage function, the cell intrinsic impact of disrupting the interaction between RAS and p110α in this central myeloid cell subset is not known.

Strengths:

Exploiting a sound previously described genetically engineered mouse model that allows tamoxifen-inducible disruption of the RAS-p110α pathway and using different readouts of macrophage activity in vitro and in vivo, the authors provide data consistent with their conclusion that alteration in RAS-p110α signaling impairs various but selective aspects of macrophage function in a cell-intrinsic manner.

Weaknesses:

My main concern is that for various readouts, the difference between wild-type and mutant macrophages in vitro or between wild-type and Pik3caRBD mice in vivo is modest, even if statistically significant. To further substantiate the extent of macrophage function alteration upon disruption of RAS-p110α signaling and its impact on the initiation and resolution of inflammatory responses, the manuscript would benefit from a more extensive assessment of macrophage activity and inflammatory responses in vivo.

In the in vivo model, all cells have disrupted RAS-p100α signaling, not only macrophages. Given that other myeloid cells besides macrophages contribute to the orchestration of inflammatory responses, it remains unclear whether the phenotype described in vivo results from impaired RAS-p100α signaling within macrophages or from defects in other haematopoietic cells such as neutrophils, dendritic cells, etc.

Inclusion of information on the absolute number of macrophages, and total immune cells (e.g. for the spleen analysis) would help determine if the reduced frequency of macrophages represents an actual difference in their total number or rather reflects a relative decrease due to an increase in the number of other/s immune cell/s.

Author response:

The following is the authors’ response to the original reviews.

Public Reviews:

Reviewer #1 (Public Review):

In this study, Alejandro Rosell et al. uncovers the immunoregulation functions of RAS-p110α pathway in macrophages, including the extravasation of monocytes from the bloodstream and subsequent lysosomal digestion. Disrupting RAS-p110α pathway by mouse genetic tools or by pharmacological intervention, hampers the inflammatory response, leading to delayed resolution and more severe acute inflammatory reactions. The authors proposed that activating p110α using small molecules could be a promising approach for treating chronic inflammation. This study provides insights into the roles and mechanisms of p110α on macrophage function and the inflammatory response, while some conclusions are still questionable because of several issues described below.

(1) Fig. 1B showed that disruption of RAS-p110α causes the decrease in the activation of NF-κB, which is a crucial transcription factor that regulates the expression of proinflammatory genes. However, the authors observed that disruption of RAS-p110α interaction results in an exacerbated inflammatory state in vivo, in both localized paw inflammation and systemic inflammatory mediator levels. Also, the authors introduced that "this disruption leads to a change in macrophage polarization, favoring a more proinflammatory M1 state" in introduction according to reference 12. The conclusions drew from the signaling and the models seemed contradictory and puzzling. Besides, it is not clear why the protein level of p65 was decreased at 10' and 30'. Was it attributed to the degradation of p65 or experimental variation?

We thank the reviewer for this insightful comment and apologize for not previously explaining the implications of the observed decrease in NF-κB activation. We found a decrease in NF-κB activation in response to LPS + IFN-γ stimulation in macrophages lacking RAS-PI3K interaction. As the reviewer pointed out, NF-κB is a key transcription factor that regulates the expression of various proinflammatory genes. To better characterize whether the decrease in p-p65 would lead to a reduction in the expression of specific cytokines, we performed a cytokine array using unstimulated and LPS + IFN-γ stimulated macrophages. The results indicated a small number of cytokines with altered expression, validating that RAS-p110α activation of p-p65 regulates the expression of some inflammatory cytokines. These results have been added to the manuscript and to Figure 1 (panels C and D). In brief, the data suggest an impairment in recruitment factors and inflammatory regulators following the disruption of RAS-p110α signaling in macrophages, which aligns with the observed in vivo phenotype.

Our findings indicate that the disruption of RAS-p110α signaling has a complex and multifaceted role in BMDMs. Specifically, monocytes lacking RAS-PI3K are unable to reach the inflamed area due to an impaired ability to extravasate, caused by altered actin cytoskeleton dynamics. Consequently, inflammation is sustained over time, continuously releasing inflammatory mediators. Moreover, we have shown that macrophages deficient in RAS-p110α interaction fail to mount a full inflammatory response due to decreased activation of p-p65, leading to reduced production of a set of inflammatory regulators. Additionally, these macrophages are unable to effectively process phagocytosed material and activate the resolutive phase of inflammation. As a result of these defects, an exacerbated and sustained inflammatory response occurs.

Our in vivo data, showing an increase in systemic inflammatory mediators, might be a consequence of the accumulation of monocytes produced by bone marrow progenitors in response to sensed inflammatory stimuli, but unable to extravasate.

Regarding the sentence in the introduction: "this disruption leads to a change in macrophage polarization, favoring a more proinflammatory M1 state" (reference 12), this was observed in an oncogenic context, which might differ from the role of RAS-p110α in a non-oncogenic situation, as analyzed in this work. We introduced these results as an example to establish the role of RAS-p110α in macrophages, demonstrating its participation in macrophage-dependent responses. Together with our study, these findings clearly indicate that p110α signaling is critical when analyzing full immune responses. Previously, little was known about the role of this PI3K isoform in immune responses. Our data, along with those presented by Murillo et al. (ref. 12), demonstrate that p110α plays a significant role in macrophage function in both oncogenic and inflammatory contexts. Additionally, our results suggest that this role is complex and multifaceted, warranting further investigation to fully understand the complexity of p110α signaling in macrophages.

Regarding decreased levels of p65 at 10’ and 30’ in RBD cells we are still uncertain about the possible molecular mechanism leading to the observed decrease. No changes in p65 mRNA levels were observed after 30 minutes of LPS+IFNγ treatment as shown in Author response image 1.

Author response image 1.

Preliminary data not shown here suggest that treating macrophages with BYL exhibits a similar effect, indicating a potential pathway for investigation. Considering that the decrease in protein levels is not due to lower mRNA expression, we may infer that post-translational mechanisms are leading to early protein degradation in RAS-p110α deficient macrophages. This could explain the observed decrease in protein activation. However, the specific molecular mechanism responsible for this degradation remains unclear, and further research is necessary to elucidate it.

(2) In Fig 3, the authors used bone-marrow derived macrophages (BMDMs) instead of isolated monocytes to evaluate the ability of monocyte transendothelial migration, which is not sufficiently convincing. In Fig. 3B, the authors evaluated the migration in Pik3caWT/- BMDMs, and Pik3caWT/WT BMDMs treated with BYL-719'. Given that the dose effect of gene expression, the best control is Pik3caWT/- BMDMs treated with BYL-719.

We thank reviewer for this comment. While we agree that using BMDMs might not be the most conventional approach for studying monocyte migration, there were several reasons why we still considered them a valid method. While isolated monocytes are the initial cell type involved in transendothelial migration, bone marrow-derived macrophages (BMDMs) provide a relevant and practical model for studying this process. BMDMs are differentiated from the same bone marrow precursors as monocytes and retain the ability to respond to chemotactic signals, adhere to endothelial cells, and migrate through the endothelium. This makes them a suitable tool for examining the cellular and molecular mechanisms underlying monocyte migration and subsequent macrophage infiltration into tissues. Additionally, BMDMs offer experimental consistency and are easier to manipulate in vitro, enabling more controlled and reproducible studies.

In response to the comment regarding Fig. 3B, we appreciate the suggestion to use Pik3ca WT/- BMDMs treated with BYL-719 as a control. However, our rationale for using Pik3ca WT/WT BMDMs treated with BYL-719 was based on a conceptual approach rather than a purely experimental control. The BYL-719 treatment in Pik3ca WT/WT cells was intended to simulate the inhibition of p110α in a fully functional, wild-type context. This allows us to directly assess the impact of p110α inhibition under normal physiological conditions, which is more representative of what would occur in an organism where the full dose of Pik3ca is present. Using Pik3ca WT/- BMDMs treated with BYL-719 as a control may not accurately reflect the in vivo scenario, where any therapeutic intervention would likely occur in the context of a fully functional, wild-type background. Our approach aims to provide a clearer understanding of how p110α inhibition affects cell functionality in a wild-type setting, which is relevant for potential therapeutic applications. Therefore, we considered the use of Pik3ca WT/WT BMDMs with BYL-719 treatment to be a more appropriate control for testing the effects of p110α inhibition in normal conditions.

(3) In Fig. 4E-4G, the authors observed that elevated levels of serine 3 phosphorylated Cofilin in Pik3caRBD/- BMDMs both in unstimulated and in proinflammatory conditions, and phosphorylation of Cofilin at Ser3 increase actin stabilization, it is not clear why disruption of RAS-p110α binding caused a decrease in the F-actin pool in unstimulated BMDMs?

We thank the reviewer for this insightful comment. During the review process, we have carefully quantified all the Western blots conducted. While we did observe an increase in phospho-Cofilin (Ser3) levels in RBD BMDMs, this increase did not reach statistical significance. As a result, we cannot confidently attribute the observed increase in F-actin to this proposed mechanism. We apologize for any confusion this may have caused. Consequently, we have removed these data from Figure 4G and the associated discussion.

Unfortunately, we have not yet identified the underlying mechanism responsible for this phenotype. Future experiments will focus on exploring potential alterations in other actin-nucleating, regulating, and stabilizing proteins that could account for the observed changes in F-actin levels.

Reviewer #2 (Public Review):

Summary:

Cell intrinsic signaling pathways controlling the function of macrophages in inflammatory processes, including in response to infection, injury or in the resolution of inflammation are incompletely understood. In this study, Rosell et al. investigate the contribution of RAS-p110α signaling to macrophage activity. p110α is a ubiquitously expressed catalytic subunit of PI3K with previously described roles in multiple biological processes including in epithelial cell growth and survival, and carcinogenesis. While previous studies have already suggested a role for RAS-p110α signaling in macrophages function, the cell intrinsic impact of disrupting the interaction between RAS and p110α in this central myeloid cell subset is not known.

Strengths:

Exploiting a sound previously described genetically mouse model that allows tamoxifen-inducible disruption of the RAS-p110α pathway and using different readouts of macrophage activity in vitro and in vivo, the authors provide data consistent with their conclusion that alteration in RAS-p110α signaling impairs the function of macrophages in a cell intrinsic manner. The study is well designed, clearly written with overall high-quality figures.

Weaknesses:

My main concern is that for many of the readouts, the difference between wild-type and mutant macrophages in vitro or between wild-type and Pik3caRBD mice in vivo is rather modest, even if statistically significant (e.g. Figure 1A, 1C, 2A, 2F, 3B, 4B, 4C). In other cases, such as for the analysis of the H&E images (Figure 1D-E, S1E), the images are not quantified, and it is hard to appreciate what the phenotype in samples from Pik3caRBD mice is or whether this is consistently observed across different animals. Also, the authors claim there is a 'notable decrease' in Akt activation but 'no discernible chance' in ERK activation based on the western blot data presented in Figure 1A. I do not think the data shown supports this conclusion.

We appreciate the reviewer's careful examination of our data and their observation regarding the modest differences between wild-type and mutant macrophages in vitro, as well as between wild-type and Pik3caRBD mice in vivo. While the differences observed in Figures 1A, 1C, 2A, 2F, 3B, 4B, and 4C are statistically significant but modest, our data demonstrate that they are biologically relevant and should be interpreted within the specific nature of our model. Our study focuses on the disruption of the RASp110α interaction, but it should be noted that alternative pathways for p110α activation, independent of RAS, remain functional in this model. Additionally, the model retains the expression of other p110 isoforms, such as p110β, p110γ, and p110δ, which are known to have significant roles in immune responses. Given the overlapping functions of these p110 isoforms, and the fact that our model involves a subtle modification that specifically affects the RAS-p110α interaction without completely abrogating p110α activity, it is understandable that only modest effects are observed in some readouts. The redundancy and compensation by other p110 isoforms likely mitigate the impact of disrupting RAS-mediated p110α activation.

However, despite these modest in vitro differences, it is crucial to highlight that the in vivo effects on inflammation are both clear and consistent. The persistence of inflammation in our model suggests that the RAS-p110α interaction plays a specific, non-redundant role in resolving inflammation, which cannot be fully compensated by other signaling pathways or p110 isoforms. These findings underscore the importance of RAS-p110α signaling in immune homeostasis and suggest that even subtle disruptions in this pathway can lead to significant physiological consequences over time, particularly in the context of inflammation. The modest differences observed may represent early or subtle alterations that could lead to more pronounced phenotypes under specific stress or stimulation conditions. This could be tested across all the figures mentioned. For instance, in Fig. 1A, the Western blot for AKT has been quantified, demonstrating a significant decrease in AKT levels; in Fig. 1C, although the difference in paw inflammation was only a few millimeters in thickness, considering the size of a mouse paw, those millimeters were very noticeable by eye. Furthermore, pathological examination of the tissue consistently showed an increase in inflammation in RBD mice. Furthermore, the consistency of the observed differences across different readouts and experimental setups reinforces the reliability and robustness of our findings. Even modest changes that are consistently observed across different assays and conditions are indicative of genuine biological effects. The statistical significance of the differences indicates that they are unlikely to be due to random variation. This statistical rigor supports the conclusion that the observed effects, albeit modest, are real and warrant further exploration.

Regarding the analysis of H&E images, we have now quantified the changes with the assistance of the pathologist, Mª Carmen García Macías, who has been added to the author list. We removed the colored arrows from the images and instead quantified fibrin and chromatin remnants as markers of inflammation staging. Loose chromatin, which increases as a consequence of cell death, is higher in the early phases of inflammation and decreases as macrophages phagocytose cell debris to initiate tissue healing. Chromatin content was scored on a scale from 1 to 3, where 1 represents the lowest amount and 3 the highest. The scoring was based on the area within the acute inflammatory abscess where chromatin could be found: 3 for less than 30%, 2 for 30-60%, and 1 for over 60%. Graphs corresponding to this quantification have now been added to Figure 1 and an explanation of the scale has been added to Material and Methods.

To further substantiate the extent of macrophage function alteration upon disruption of RAS-p110α signaling, the manuscript would benefit from testing macrophage activity in vitro and in vivo across other key macrophage activities such as bacteria phagocytosis, cytokine/chemokine production in response to titrating amounts of different PAMPs, inflammasome function, etc. This would be generally important overall but also useful to determine whether the defects in monocyte motility or macrophage lysosomal function are selectively controlled downstream of RAS-p110α signaling.

We thank reviewer #2 for this comment. In order to better address the role of RAS-PI3K in macrophage function, we have performed some additional experiments, some of which have been added to the revised version of the manuscript.

(1) We have performed cytokine microarrays of RAS-p110α deficient macrophages unstimulated and stimulated with LPS+IFN-g. Results have been added to the manuscript and to Supplementary Figure S1E and S1F. In brief, the data obtained suggest an impairment in recruitment factors, as well as in inflammatory regulators after disruption of RAS-p110α signaling in macrophages, which align with the in vivo observed phenotype.

(2) We also conducted phagocytosis assays to analyze the ability of RAS-p110α deficient macrophages to phagocytose 1 µm Sepharose beads, Borrelia burgdorferi, and apoptotic cells. The data reveal varied behavior of RAS-p110α deficient bone marrow-derived macrophages (BMDMs) depending on the target:

• Engulfment of Non-biological Particles: RAS-p110α deficient macrophages showed a decreased ability to engulf 1 µm Sepharose beads. This suggests that RAS-p110α signaling is important for the effective phagocytosis of non-biological particles. These findings have now been added to the text and figures have been added to supplementary Fig. S4A

• Response to Bacterial Pathogens: When exposed to Borrelia burgdorferi, RAS-p110α deficient macrophages did not exhibit a change in bacterial uptake. This indicates that RAS-p110α may not play a critical role in the initial phagocytosis of this bacterial pathogen. The observed increase in the phagocytic index, although not statistically significant, might imply a compensatory mechanism or a more complex interaction that warrants further investigation. These findings have now been added to the text and figures have been added to supplementary Fig. S4B. These experiments were performed in collaboration with Dr. Anguita, from CICBioBune (Bilbao, Spain) and, as a consequence, he has been added as an author in the paper.

• Phagocytosis of Apoptotic Cells: There were no differences in the phagocytosis rate of apoptotic cells between RAS-p110α deficient and control macrophages at early time points. However, the accumulation of engulfed material at later time points suggests a possible delay in the processing and degradation of apoptotic cells in the absence of RAS-p110α signaling.

These findings highlight the complexity of RAS-p110α's involvement in phagocytic processes and suggest that its role may vary with different types of phagocytic targets.

Furthermore, given the key role of other myeloid cells besides macrophages in inflammation and immunity it remains unclear whether the phenotype observed in vivo can be attributed to impaired macrophage function. Is the function of neutrophils, dendritic cells or other key innate immune cells not affected?

Thank you for this insightful comment. We understand the key role of other myeloid cells in inflammation and immunity. However, our study specifically focuses on the role of macrophages. Our data show that disruption of RAS-PI3K leads to a clear defect in macrophage extravasation, and our in vitro data demonstrate issues in macrophage cytoskeleton and phagocytosis, aligning with the in vivo phenotype.

Experiments investigating the role of RAS-PI3K in neutrophils, dendritic cells, or other innate immune cells are beyond the scope of this study. Understanding these interactions would indeed require separate, comprehensive studies and the generation of new mouse models to disrupt RAS-PI3K exclusively in specific cell types.

Furthermore, during paw inflammation experiments, polymorphonuclear cells were present from the initial phases of the inflammatory response. What caught our attention was the prolonged presence of these cells. In conversation with our in-house pathologist, she mentioned the lack of macrophages to remove dead polymorphonuclear cells in our RAS-PI3K mutant mice. Specific staining for macrophages confirmed the absence of macrophages in the inflamed node of mutant mice.

We acknowledge that further research is necessary to elucidate the effects on other myeloid cells. However, our current findings provide clear evidence of a decrease in inflammatory monocytes and defective macrophage responses to inflammation, both in vivo and in vitro. We believe these results significantly contribute to understanding the role of RAS-PI3K in macrophage function during inflammation.

Compelling proof of concept data that targeting RAS-p110α signalling constitutes indeed a putative approach for modulation of chronic inflammation is lacking. Addressing this further would increase the conceptual advance of the manuscript and provide extra support to the authors' suggestion that p110α inhibition or activation constitute promising approaches to manage inflammation.

We thank Reviewer #2 for this insightful comment. In our manuscript, we have demonstrated through multiple experiments that the inhibition of p110α, either by disrupting RAS-p110α signaling or through the use of Alpelisib (BYL-719), has a modulatory effect on inflammatory responses. However, we acknowledge that we have not activated the pathway due to the unavailability of a suitable p110α activator until the concluding phase of our study.

We recognize the importance of this point and are eager about investigating both the inhibition and activation of p110α as potential approaches to managing inflammation in well-established inflammatory disease models. We believe that such comprehensive studies would significantly enhance the conceptual advance and translational relevance of our findings.

However, it is essential to note that the primary aim of our current work was to demonstrate the role of RAS-p110α in the inflammatory responses of macrophages. We have successfully shown that RASp110α influences macrophage behavior and inflammatory signaling. Expanding the scope to include disease models and pathway activation studies would be an extensive project that goes beyond the current objectives of this manuscript. While our present study establishes the foundational role of RASp110α in macrophage-mediated inflammatory responses, we agree that further investigation into both p110α inhibition and activation in disease models is crucial. We are keen to pursue this line of research in future studies, which we believe will provide robust evidence supporting the therapeutic potential of targeting RAS-p110α signaling in chronic inflammation.

Finally, the analysis by FACS should also include information about the total number of cells, not just the percentage, which is affected by the relative change in other populations. On this point, Figure S2B shows a substantial, albeit not significant (with less number of mice analysed), increase in the percentage of CD3+ cells. Is there an increase in the absolute number of T cells or does this apparent relative increase reflect a reduction in myeloid cells?

We thank the reviewer for this comment, which we have addressed in the revised version of the manuscript. Regarding the total number of cells analyzed, we have added to the Materials and Methods section that in all our studies, a total of 50,000 cells were analyzed (line 749). The percentages of cells are related to these 50,000 events. Additionally, we have increased the number of mice analyzed by including new mice for CD3+ cell analysis. Despite this, the results remain not significant.

Recommendations for the authors:

Reviewer #1 (Recommendations For The Authors):

(1) It is recommended to provide a graphical abstract to summarize the multiple functions of RAS-p110α pathway in monocyte/macrophages that the authors proposed

We thank reviewer for this useful recommendation. A graphical abstract has now been added to the study.

(2) Western blots in this paper need quantification and a measure of reproducibility

We have now added a graph with the quantification of the western blots performed in this work as a measure of reproducibility.

(3) Representative flow data and gating strategy should be included

We have now added the description of the gating strategy followed to material and methods section.