Activation of the P2RX7/IL-18 pathway in immune cells attenuates lung fibrosis

Reviewer #1 (Public Review):

In this study the authors investigate whether a presumably allosteric P2RX7 activating compound that they previously discovered reduces fibrosis in a bleomycin mouse model. They chose this particular model as publicly available mRNA data indicate that the P2XR7 pathway is downregulated in idiopathic pulmonary fibrosis patients compared to control individuals. The authors first demonstrate that two proxies of lung damage, Ashcroft score and collagen fibers, are significantly reduced in the bleomycin model on HEI3090 treatment. Additional data implicate specific immune cell infiltrates and cytokines, namely inflammatory macrophages and damped release of IL-17A, as potential mechanistic links between their compound and reduced fibrosis. Finally, the researchers transplant splenocytes from WT, NLRP3-KO, and IL-18-KO mice into animals lacking the P2XR7 receptor to specifically ascertain how the transplanted splenocytes, which are WT for P2XR7 receptor, respond to HEI3090 (a P2XR7 agonist). Based on these results, the authors conclude that HEI3090 enhanced IL-18 production through the P2XR7-NLRP3 inflammasome axis to dampen fibrosis.

These findings could be interesting to the field, as there are conflicting results as to whether NLRP3 activation contributes to fibrosis and if so, at what stage(s) (e.g., acute damage phase versus progression). However, major weaknesses of the study include inconsistent and small effect sizes in key outcomes used to measure fibrosis, small animal cohorts that do not empower results, and lack of key experimental controls. For example, damage indicators for the vehicle-treated mice transplanted with splenocytes of various genetic background are markedly different, and there are no statistical tests of these effects because the data are presented as separate graphs. Moreover, the fundamental assumption that HEI3090 acts specifically through the P2XR7 pathway in this model is questionable, as P2XR7 knockout mice are not included in the initial key experiments. These issues must be addressed as stimulating an inflammasome response might lead to pathogenic inflammation, which could counterproductively exacerbate fibrosis in the clinic and harm people.

Experimental concerns:

1. Ashcroft method quantification throughout is outdated and prone to bias. The methods describing quantification are lacking, and only include a citation: there should be mention of researcher blinding, etc. In general, please re-quantify using an automated classifier, and consider staining for additional markers of lung damage that are appropriate in the field.

2. For Figure 2, P2XR7 knockout mice, and an additional P2XR7 activator, should be included (e.g, A74003, AZ10606120, others), to support the hypothesis that HEI3090 acts through this pathway to alleviate fibrosis. Moreover, these data are especially important as the author's conclusions are directly opposed to a previous study demonstrating that the P2XR7 receptor is required for inflammation/fibrosis in this model system (PMID: 20522787). Two-way ANOVA or similar statistical tests on all groups should be examined to see whether genetic knockout of this DAMP receptor alone is protective or exacerbates fibrosis (e.g., comparing the vehicle-alone groups), and whether compounds exert a specific effect through this receptor.

3. Fig. 3A: Please show the individual IFN/IL-17A plots in the supplement, as a ratiometric result might mask variance. Moreover, please conduct a statistical test for the outlier in the HEI3090 condition (to potentially remove it), as this sole data point might skew the entire mean, causing the observed statistical difference between means despite a very modest change. If the results are still significant, please comment on effect size.

4. Fig. 3: How is IL-17A measured and what is the abbreviation GMFI?

5. Fig. 3E: It's unclear how the left and right figures align-it looks like the gates are 45.8 % and 25 %, respectively, but the means on the right are between 2-3%. Also, is this effect size (2 versus 3 %) significant biologically?

6. For Figure 4B-G, the Ashcroft scores for the vehicle mice treated with HEI3090 are at entirely different starting points following adoptive transfer of cells with different genetic background. In Fig. 1, WT mice have starting scores of around 3 following the induction of fibrosis, with a modest decrease of about 0.8 following HEI3090 treatment. Here, there is a much greater effect of the genetic background itself rather than the treatment, with the IL-18 knockout mice having a much lower baseline "vehicle" score (~1) compared to Fig. 1F (both of which are 14 day treatments). In fact, adoptive transfer of WT splenocytes start at a baseline of 1.8 here, which is much lower than Fig. 1F, and NLRP3-KO splenocytes score nearly the same as Fig. 1F following BLM treatment, with a modest reduction following treatment with HEI3090. Please analyze all of these groups together with appropriate multiple hypothesis testing to examine the effect of the genetic background, and please comment on why IL-18-knockout splenocytes might be protective at vehicle baseline while NLRP3-knockout splenocytes might exacerbate the phenotype at vehicle baseline.

7. The variance on Supplemental Figure 5C is quite large. These data have a decrease in mean Ashcroft score between untreated and HEI3090 treatment of around 0.8, which is similar to the WT mice in Figure 1. This is very concerning, as the underlying assumption is that KO of the protein required for HEI3090's on-target effect would completely ablate response, and this would be required for the subsequent adoptive transfer experiments in Figure 4. Please conduct power analysis, comment, and provide additional evidence (other than Ashcroft score).

8. Figure 4: Should quantify collagen fibers or have an additional quantitative metric for lung damage, as in Fig. 2C/J.

9. Figure 4: Should group the quantification of C/E/G and perform a 2-way Anova to assess effects of genetic background versus treatment.

10. Fig. 4H, Supplemental Fig. 6D: Is it reasonable to expect differences in IL-1beta and IL-18 in sera compared to in lung tissue itself?

Reviewer #2 (Public Review):

In the study by Hreich et al, the potency of P2RX7 positive modulator HEI3090, developed by the authors, for the treatment of Idiopathic pulmonary fibrosis (IPF) was investigated. Recently, the authors have shown that HEI3090 can protect against lung cancer by stimulating dendritic cell P2RX7, resulting in IL-18 production that stimulates IFN-γ production by T and NK cells (DOI: 10.1038/s41467-021-20912-2). Interestingly, HEI3090 increases IL-18 levels only in the presence of high eATP. Since the treatment options for IPF are limited, new therapeutic strategies and targets are needed. The authors first show that P2RX7/IL-18/IFNG axis is downregulated in patients with IPF. Next, they used a bleomycin-induced lung fibrosis mouse model to show that the use of a positive modulator of P2RX7 leads to the activation of the P2RX7/IL-18 axis in immune cells that limits lung fibrosis onset or progression. Mechanistically, treatment with HEI3090 enhanced IL-18-dependent IFN-γ production by lung T cells leading to a decreased production of IL-17 and TGFβ, major drivers of IPF. The major novelty is the use of the small molecule HEI3090 to stimulate the immune system to limit lung fibrosis progression by targeting the P2RX7, which could be potentially combined with current therapies available. However, there is the lack of information on the reproducibility of data, especially for the data presented in Figures 3 and 4, and related supplementary figures, as well as the lack of support data for experiments that emphasize the role of P2RX7 expressed on immune cells (e.g. frequency of transferred cells compared to endogenous cells).

Reviewer #3 (Public Review):

Idiopathic pulmonary fibrosis (IPF) is an aggressive interstitial lung disease with progressive and irreversible deterioration of respiratory functions that lacks curative therapies. The authors investigated a new therapeutic approach to treat idiopathic pulmonary fibrosis by targeting P2RX7/IL-18/IFNG axis.

The current data are mainly based on P2RX7 activator HEI3090 and genetic experiments are lacking to support the primary claim that activation P2RX7/IL-18/IFNG axis is beneficial for IPF.

- Parenteral systemic administration of IFN-γ failed in clinical trials (INSPIRE; NCT00075998). However, this study used i.p. administration of P2RX7 activator HEI3090 to activate P2RX7/IL-18/IFNG axis.

- Activation of P2RX7 NLRP3 inflammasome triggers cell death and the current experiments do not explore IL-18 as a potential therapy that would avoid harmful cell death as a consequence of P2RX7/NLRP3 inflammasome activation.

- Reciprocal bone marrow chimera model is needed to demonstrate the requirement of a hematopoietic compartment for HEI3090's antifibrotic effect.

- There is no evidence to show whether P2RX7 interferes with bleomycin during the generation of the IPF model. Independent IPF models would validate the therapeutic effect of P2RX7.

Author Response

eLife assessment

This study presents a potentially valuable discovery which indicates that activation of the P2RX7 pathway can reduce the degree of lung fibrosis caused by other inflammatory pathways. If confirmed, the study could clarify the role of specific immune networks in the establishment and progression of lung fibrosis.

Thanks for this positive comment. Indeed, knowing that lung fibrosis is partly driven by inflammation, with a dysregulated Th1/Th2/Th17 ratio (PMID 20176803, PMID 19682929), we hypothesized that modulating the immune response would be able to attenuate lung fibrosis. To address this issue, we proposed to boost the activation of P2RX7, a purinergic receptor with immunomodulatory properties (PMID 8614837, PMID 11035104), in the well characterized bleomycin-induced lung fibrosis mouse model (PMID 25959210). In this study, we used a pyroglutamic derivative compound (HEI3090) able to specifically enhance P2RX7-dependent biological activities (cationic channel and macropore opening) only in the presence of extracellular ATP, which was qualified as the first representative of an immunotherapy relying on the activation of P2RX7 expressed by dendritic cells (PMID 33510147), and we showed that lung fibrosis is attenuated in mice treated with HEI3090 as compared to vehicle treated mice.

However, the presented data and analyses are incomplete as they rely on limited pharmacological treatments and because there is an absence of key control studies, validation experiments and statistical analyses.

Quantification of lung fibrosis:

Quantification of lung fibrosis was made on the basis of a modified Ashcroft score which assigns 8 grades to quantify lung fibrosis reliably and reproducibly (PMID 18476815). To be even more accurate and not biased by patchy lesions observed in all existing lung fibrosis induced mouse models, the whole lungs (left and right lobes) were divided in section of 880 µm2 and each section was scored individually. A total of 80 to 110 sections were analyzed per mouse. We agree that our text requires clarification. In parallel, the collagen amount given by the polarization intensity of the Sirius red staining of the lung slices was quantified with a homemade ImageJ/Fiji macro program. Further, we recently analyzed by FACS the percentage of PDGFRα (a specific marker of fibroblasts and myofibroblasts) positive cells in lungs isolated from vehicle and HEI3090-treated mice. All these 3 different markers of lung damage show that HEI3090 attenuates bleomycin-induced lung fibrosis and therefore validate the use of the Ashcroft score to accurately study the extend of lung fibrosis. We are going add quantification of collagen fibers in all figures.

Limited pharmacological treatments:

We have designed and characterized HEI3090 in a previous study and have shown that it is a positive modulator of P2RX7 (PMID 33510147).

To test its effect on lung fibrosis, we tested two pharmacological regimens using HEI3090 and have shown that both regimens are effective in limiting the progression of fibrosis. While having shown the requirement of P2RX7 for the activity of HEI3090 (PMID 33510147), we used in this study p2rx7 KO mice which were adoptively transferred with splenocytes isolated from p2rx7 KO mice to demonstrate the involvement of P2RX7 to mediate the antifibrotic effect of HEI3090. This experiment also serves as control to validate the adoptive transfer experiment.

We agree that proving and validating furthermore that activation of the P2RX7/IL-18 pathway can limit the progression of fibrosis requires the use of other activators of P2RX7. However, to date, HEI3090 is the only pharmacological compound described to activate the receptor. Indeed, the other chemical compounds described in the literature are negative allosteric modulator of P2RX7 (PMID 27935479),

Absence of key control studies and validation experiments:

The importance of P2RX7 in the antifibrotic effect of HEI3090 was demonstrated thanks to P2RX7 KO mice (supplementary figures S6B). We are going to implement this figure with additional mice.

The importance of immune cells was demonstrated thanks to adoptive transfer of WT splenocytes (expressing P2RX7) into P2RX7 KO mice. We agree that lung fibrosis is attenuated in vehicle-treated P2RX7 KO mice, but lung fibrosis is still present and could be modulated by treatments as demonstrated by adoptive transfer of splenocytes isolated from IL-1B KO mice who still respond to HEI3090 as shown in Supplementary figure S6C.

As suggested by reviewers we examined the effect of genetic background using two-way Anova test and the result is “the interaction is considered not significant”.

The prevalence of transferred immune cells on endogenous cells is demonstrated in supplementary figure S5, where intravenous injection of splenocytes isolated from P2RX7 KO mice into WT mice abolishes the antifibrotic effect of HEI3090. This experiment further validates the requirement of immune cells and the efficacy of the adoptive transfer approach.

Statistical analyses:

In this study we compared side by side the effect of HEI3090 versus vehicle in different genetic backgrounds in order to characterize the implication of actors of the P2RX7/IL-18 pathway in the antifibrotic effect of HEI3090. We also examined the effect of genetic background using the two-way Anova test. Following European recommendations, and in agreement with the ARRIVE guidelines for mice studies, we performed provisional statistic to evaluate the number of mice required in the study and stopped the experiments when significantly statistical results were observed. We agree that results are heterogeneous, however this heterogeneity does not prevent data analyses as shown in supplementary figure S6D, where adoptive transfer of splenocytes isolated from IL-1B KO mice into P2RX7 KO mice dampens BLM-induced lung fibrosis (with an Ashcroft score of 1.8 versus 3 in WT mice) but still responds to HEI3090, thus indicating that IL-1B is not required to mediate the antifibrotic effect of HEI3090.