Chemotherapy activates inflammasomes to cause inflammation-associated bone loss

Reviewer #1 (Public Review):

Summary:

Doxorubincin has long been known to cause bone loss by increasing osteoclast and suppressing osteoblast activities. The study by Wang et al. reports a comprehensive investigation into the off-target effects of doxorubicin on bone tissues and potential mechanisms.. They used a tumor-free model with wild type mice and found that even a single dose of doxorubicin has a major influence by increasing leukopenia and DAMPs and inflammasomes in macrophages and neutrophils, and inflammation-related cell death (pyroptosis and NETosis). The gene knockout study shows that AIM2 and NLRP3 are the major contributors to bone loss. Overall, the study confirmed previous findings regarding the impact of doxorubicin on tissue inflammation and expands the research further into bone tissue. The presented data presented are consistent; however, a major question remains regarding whether doxorubicin drives inflammation and its related events. Most in vitro study showed that the effect of doxorubincin cannot be demonstrated without LPS priming. This observation raises the question of whether doxorubincin itself could activate the inflammasome and the related events. In vivo study, on the other hand, suggested that it doesn't require LPS. The inconsistency here was not explained further. Moreover, a tumor-free mouse model was used for the study; however, immune responses in tumor bearing models would likely be distinct from tumor-free ones. The justification for using tumor-free models is not well-established.

Strengths:
The paper includes a comprehensive study that shows the effects of doxorubincin on cytokine levels in serum, release of DAMPs and NETosis, and leukopenia using both in vivo and in vitro models. Bone marrow cells, macrophages and neutrophils were isolated from the bone marrow, and the levels of cytokines in serum were also determined.

They employed multiple knockout models with deficiency in Aim 2, Nlirp3, and double deficiencies to dissect the functional involvement of these two inflammasomes.

The experiments in general are well designed. The paper is also logically written, and figures were clearly labeled.

Weaknesses:
Most of the data presented are correlative, and there is not much effort to dissect the underlying molecular mechanism.

It is not entirely clear why a tumor free model is chosen to study immune responses, as immune responses can differ significantly with or without tumor-bearing.

Immune responses in isolated macrophages, neutrophils and bone marrow cells require priming with LPS, while such responses are not observed in vivo. There is no explanation for these differences.

The band intensities on Western blots in Fig. 4 and Fig. 5 are not quantified, and the numbers of repeats are also not provided.

Many abbreviations are used throughout the text, and some of the full names are not provided.

Fig. 5B needs a label on X axis.

Reviewer #2 (Public Review):

Summary:

Wang and collaborators have evaluated the impact of inflammation on bone loss induced by Doxorubicin, which is commonly used in chemotherapy to treat various cancers. In mice, they show that a single injection of Doxorubicin induces systemic inflammation, leukopenia, and a significant bone loss associated with increased bone-resorbing osteoclast numbers. In vitro, the authors show that Doxorubicin activates the AIM2 and NLRP3 inflammasomes in macrophages and neutrophils. Importantly, they show that the full knockouts (germline deletions) of AIM2 (Aim2-/-) and NLRP3 (Nlrp3-/-) and Caspase 1 (Casp1-/-) limit (but do not completely abolish) bone loss induced 4 weeks after a single injection of Doxorubicin in mice. From these results, they conclude that Doxorubicin activates inflammasomes to cause inflammation-associated bone loss.

Strength:

This manuscript provides functional experiments demonstrating that NRLP3 and/or AIM2 loss-of-functions (and thus the systemic impairment of the inflammatory response) prevent bone-loss induced by Doxorubicin in mice.

Weaknesses:

Numerous studies have reported that Doxorubicin induces systemic inflammation and activates the inflammasome in myeloid cells and various other cell types. It is also known that systemic inflammation and Doxorubicin treatment lead to bone loss. Hence, the key conclusions drawn from this work have been known already or were very much expected. Therefore, the novelty appears somewhat limited. One important limitation is the lack of experiments that could determine which cell lineages are involved in bone loss induced by Doxorubicin in vivo, while the tools to do so exist. The characterization of the bone phenotype is incomplete, and unfortunately does not tell us whether the inflammasome is activated in some of the cell lineages present in bones in vivo. Another limitation is that the relative importance of the inflammasomes compared to cell senescence and autophagy, which are also induced by Doxorubicin, has not been evaluated. Hence the main molecular mechanisms responsible for bone loss induced by Doxorubicin in vivo remains unknown. Lastly, it would have been interesting, on a more clinical point of view, to compare the few relevant treatments that could limit the deleterious effect of Doxorubicin on bone loss while preserving the toxicity on tumor cells.

Author Response

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

Reviewer #1:

(1) It is not entirely clear why a tumor-free model is chosen to study immune responses, as immune responses can differ significantly with or without tumor-bearing. A more detailed explanation is needed.

We appreciate the question. As stated in the original submission, tumor-free mouse models are commonly used to assess off-target outcomes of anti-neoplastic therapies. We have expanded on this point and acknowledged this shortcoming in the revised manuscript (lines 264-265).

(2) Immune responses in isolated macrophages, neutrophils, and bone marrow cells require priming with LPS, while such responses are not observed in vivo. There is no explanation for these differences.

The reviewer raises an excellent point. The assembly of inflammasomes such as those nucleated by NLRP3 requires priming signals, which increase the levels of this sensor, which are kept low in homeostatic conditions to prevent spontaneous unwanted inflammation. While LPS is commonly used in vitro as an inducer of priming signals, these cues are triggered in vivo by various molecules, including pro-inflammatory cytokines. We have provided a rationale for the use of LPS in vitro in the revised manuscript (lines 144-145).

(3) The band intensities on Western blots in Fig. 4 and Fig. 5 are not quantified, and the numbers of repeats are also not provided. This additional information is recommended.

While caspase-1, caspase-3, GSDMD, and GSDME but not AIM 2 and NLRP3 are activated upon proteolytic cleavage. It is not straightforward to quantify and describe the intensity of the bands of these numerous with different fate outcomes. We regret for not mentioning the numbers of repeats in the original submission. This information has now been provided in figure legends where necessary.

(4) Many abbreviations are used throughout the text, and some of the full names are not provided.

Full names are required at the first introduction.

We agree. We have provided full names at the first introduction (lines 21, 23, 86).

(5) Fig. 5B needs a label on the X axis.

We regret the confusion: X axis was for both Fig. 5B and 5C. We have made the change in the new Fig. 5.

Reviewer #2:

The following specific points could be addressed to further improve the quality of the manuscript:

(1) Concerning data presented in Figure 1, 3D micro-CT reconstructions of the entire femurs could be shown instead of just the trabecular bone. Data on cortical bone loss are important. It would be important to show histological (sagittal) sections of the bones at baseline, treated with Doxorubicin or vehicle, and quantify osteoblasts in addition to osteoclasts. Is there increased bone marrow adiposity in Doxorubicin-treated mice? The data with vehicle should be shown in the main figures not just in the supplemental data.

We thank the reviewer for the suggestion. We have now provided 3 D micro-CT reconstructions of a representative femur containing both trabecular and cortical bones (S1B Fig). Only the metaphyseal area is shown because we did not originally scan the entire femur.

Quantification of osteoblast number is not a reliable measurement, the reason why we carried out dynamic histomorphometry to assess the effect of doxorubicin on bone formation (original S1D Fig/new S1E Fig).

Unfortunately, we did not determine the effects of doxorubicin on bone marrow adiposity. However, to address the reviewer’s comment, we have mentioned in the revised manuscript adipogenic effects of doxorubicin based on the literature (lines 264-265).

(2) Concerning data presented in Figure 2, how long after Doxorubicin injection is leukopenia observed (beyond the 72-hour timepoint)? Does cell-count return to baseline 4 weeks after treatment (when the bone phenotype is characterized)? Why use 12-week-old mice here and 10week-old animals for the rest of the study?

We appreciate the question. We did not measure leukopenic effects of doxorubicin beyond the 72-hour timepoint based on the following: i) bones are analyzed in mice injected only once with a single dose of doxorubicin; ii) leukopenia is a side effect of doxorubicin whose blood levels should be undetectable 4 weeks after its administration although we did not measure them experimentally. Our premise is that osteopenia observed in doxorubicin-exposed mice is the result of early events that occur after the administration of the drug.

We apologize for the confusion. We assessed baseline bone mass by VivaCT using 10-week-old mice; doxorubicin was injected 2 weeks when mice were 12-week-old. We have clarified this point in the revised manuscript (line 301).

(3) It would be important to evaluate local inflammation in bones collected from wild-type and mutant mice. Are ASC specks, Cit-H3, and MPO present in the bone marrow? The expression of some components of the inflammasomes or relevant pathways could be assessed in bone samples deprived of bone marrow and in the bone marrow.

This is a good point. Although we were not able to reliably measure Cit-H3 and MPO in bone marrow fluid, our data shown in Figs. 3-6, 7A-D are from bone marrow cells.

(4) Data presented in western blots should be quantified. The ratio of signal intensity obtained for beta-actin over the signal obtained for a given protein should be calculated for each experimental condition (especially in Figure 5, where beta-actin levels fluctuate a lot).

Please see the response to question #1. Fluctuations in β-actin levels are likely related to doxorubicin cytotoxic effects as mentioned in the original submission (lines 150, 194, 253). Despite this caveat, IL-1β levels are stimulated by this drug.

(5) In Figure 7, BV/TV of WT and mutant mice at baseline should be quantified and shown. Sagittal histological sections of the femur should be shown. 3D micro-CT reconstructions of the entire femur could be shown instead of just the trabecular bone. Osteoblasts and bone resorption should be quantified. Data obtained with vehicle should be quantified and shown in the main figure. The control and LPS conditions should be better defined. Does it include vehicle?

Please see the response to reviewer 1’s question #1.

We have now provided 3 D micro-CT reconstructions of a representative femur containing both trabecular and cortical bone (S3A, B Fig).

LPS was dissolved in PBS (vehicle), which was used as control. We have now replaced vehicle with PBS in Fig. 7.

(6) For all figures, the number of biological replicates should be mentioned in the legends, as well as the statistical tests used for the analyses.

We have now included this information in the legends where necessary.

(7) Some of the scientific rationales are not totally clear and could be better explained in the text. For example, it is written on page 6 "studies mainly on male mice and revolved around innate immune responses" and "we focused on neutrophils because of their high turnover rate and short lifespan", but it is not clear why. The rationale (page 10) for assessing bone mass in "mice globally lacking AIM2 and/or NLRP3" is not totally clear either. The argument is that systemic inflammation leads to bone loss but the effects obtained with the total ablation of AIM2 and NLRP3 do not prove strictly speaking that systemic inflammation really matters (in this current study, although we know from many other studies that it clearly does matter). We could imagine, for example, that bone mass would be preserved in AIM2 KO mice only because the inflammasome is impaired in osteoblasts and/or osteoclasts, but not in any other cell types. Conversely one could imagine that bone would be preserved only because inflammation is preserved in the gut, for example. The use of global knockouts unfortunately does not tell us much about the importance of systemic versus local effects of the inflammasomes. It shows that reducing inflammation, either in specific organs or globally, limits bone loss in doxorubicin-treated mice. This result is important but it was fully expected since doxorubicin has been reported to induce systemic inflammation, and since many studies have shown that systemic inflammation leads to bone loss.

We appreciate the comments. We have clarified the rationale for focusing on neutrophils (lines 129-130) and AIM2 and NLRP inflammasomes (lines 209-211). We have also now down played the concept of inflammasome-mediated systemic inflammation in doxorubicin-induced bone loss.