Ym1 protein crystals promote type 2 immunity

Reviewer #1 (Public Review):

Summary: The manuscript by Heyndrickx et al describes protein crystal formation and function that bears similarity to Charcot-Leyden crystals made of galectin 10, found in humans under similar conditions. Therefore, the authors set out to investigate CLP crystal formation and their immunological effects in the lung. The authors reveal the crystal structure of both Ym1 and Ym2 and show that Ym1 crystals trigger innate immunity, activated dendritic cells in the lymph node, enhancing antigen uptake and migration to the lung, ultimately leading to induction of type 2 immunity.

Strengths:
We know a lot about expression levels of CLPs in various settings in the mouse but still know very little about the functions of these proteins, especially in light of their ability to form crystal structures. As such data presented in this paper is a major advance to the field.

Resolving the crystal structure of Ym2 and the comparison between native and recombinant CLP crystals is a strength of this manuscript that will be a very powerful tool for further evaluation and understanding of receptor, binding partner studies including the ability to aid mutant protein generation.

The ability to recombinantly generate CLP crystals and study their function in vivo and ex vivo has provided a robust dataset whereby CLPs can activate innate immune responses, aid activation and trafficking of antigen presenting cells from the lymph node to the lung and further enhances type 2 immunity. By demonstrating these effects the authors directly address the aims for the study. A key point of this study is the generation of a model in which crystal formation/function an important feature of human eosinophilic diseases, can be studied utilising mouse models. Excitingly, using crystal structures combined with understanding the biochemistry of these proteins will provide a potential avenue whereby inhibitors could be used to dissolve or prevent crystal formation in vivo.

The data presented flows logically and formulates a well constructed overall picture of exactly what CLP crystals could be doing in an inflammatory setting in vivo. This leaves open a clear and exciting future avenue (currently beyond the scope of this work) for determining whether targeting crystal formation in vivo could limit pathology.

Weaknesses:
Although resolving the crystal structure of Ym2 in particular is a strength of the authors work, the weaknesses are that further work or even discussion of Ym2 versus Ym1 has not been directly demonstrated. The authors suggest Ym2 crystals will likely function the same as Ym1, but there is insufficient discussion (or data) beyond sequence similarity as to why this is the case. If Ym1 and Ym2 crystals function the same way, from an evolutionary point, why do mice express two very similar proteins that are expressed under similar conditions that can both crystalise and as the authors suggest act in a similar way. Some discussion around these points would add further value.

Additionally, the crystal structure for Ym1 has been previously resolved (Tsai et al 2004, PMID 15522777) and it is unclear whether the data from the authors represents an advance in the 3D structure from what is previously known.

Whilst also generating a model to understand charcot-leyden crystals (CLCs), the authors fail to discuss whether crystal shape may be an important feature of crystal function. CLCs are typically needle like, and previous publications have shown using histology and TEM that Ym1 crystals are also needle like. However, the crystals presented in this paper show only formation of plate like structures. It is unclear whether these differences represent different methodologies (ie histology is 2D slides), or differences in CLP crystals that are intracellular versus extracellular. These findings highlight a key question over whether crystal shape could be important for function and has not been addressed by the authors.

Ym1/Ym2 crystals are often observed in conditions where strong eosinophilic inflammation is present. However, soluble Ym1 delivery in naïve mice shows crystal formation in the absence of a strong immune response. There is no clear discussion as to the conditions in which crystal formation occurs in vivo and how results presented in the paper in terms of priming or exacerbating an immune responses align with what is known about situations where Ym1 and Ym2 crystals have been observed.

Reviewer #2 (Public Review):

Summary: This interesting study addresses the ability of Ym1 protein crystals to promote pulmonary type 2 inflammation in vivo, in mice.

Strengths: The data are extremely high quality, clearly presented, significantly extending previous work from this group on the type 2 immunogenicity of protein crystals.

Weaknesses: There are no major weaknesses in this study. It would be interesting to see if Ym2 crystals behave similarly to Ym1 crystals in vivo. Some additional text in the Introduction and Discussion would enrich those sections.