RAS-p110α signalling in macrophages is required for effective inflammatory response and resolution of inflammation

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    This useful study investigates the impact of disrupting the interaction of RAS with the PI3K subunit p110α in macrophage function in vitro and inflammatory responses in vivo. Solid data overall supports a role for RAS-p110α signalling in regulating macrophage activity and so inflammation, however for many of the readouts presented the magnitude of the phenotype is not particularly pronounced. Further analysis would be required to substantiate the claims that RAS-p110α signalling plays a key role in macrophage function. Of note, the molecular mechanisms of how exactly p110α regulating the functions in macrophage have not yet been established.

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Abstract

Macrophages are crucial in the body's inflammatory response, with tightly regulated functions for optimal immune system performance. Our study reveals that the RAS-p110α signalling pathway, known for its involvement in various biological processes and tumorigenesis, regulates two vital aspects of the inflammatory response in macrophages: the initial monocyte movement and later-stage lysosomal function. Disrupting this pathway, either in a mouse model or through drug intervention, hampers the inflammatory response, leading to delayed resolution and the development of more severe acute inflammatory reactions in live models. This discovery uncovers a previously unknown role of the p110α isoform in immune regulation within macrophages, offering insight into the complex mechanisms governing their function during inflammation. With emerging potential to activate p110α using small molecules, targeting the RAS-p110α pathway could be a promising approach for treating chronic inflammation. This therapeutic prospect holds significant promise for easing inflammatory disorders and improving the quality of life for affected patients.

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  1. eLife assessment

    This useful study investigates the impact of disrupting the interaction of RAS with the PI3K subunit p110α in macrophage function in vitro and inflammatory responses in vivo. Solid data overall supports a role for RAS-p110α signalling in regulating macrophage activity and so inflammation, however for many of the readouts presented the magnitude of the phenotype is not particularly pronounced. Further analysis would be required to substantiate the claims that RAS-p110α signalling plays a key role in macrophage function. Of note, the molecular mechanisms of how exactly p110α regulating the functions in macrophage have not yet been established.

  2. 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, favouring 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?

    (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.

    (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?

  3. 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.

    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.

    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?

    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.

    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?