Immunoglobulin M regulates airway hyperresponsiveness independent of T helper 2 allergic inflammation

Reviewer #1 (Public Review):

Summary: The authors of this study sought to define a role for IgM in responses to house dust mites in the lung.

Strengths:

Unexpected observation about IgM biology
Combination of experiments to elucidate function

Weaknesses:

Would love more connection to human disease

Reviewer #2 (Public Review):

Summary:
The manuscript by Hadebe and colleagues describes a striking reduction in airway hyperresponsiveness in Igm-deficient mice in response to HDM, OVA and papain across the B6 and BALB-c backgrounds. The authors suggest that the deficit is not due to improper type 2 immune responses, nor an aberrant B cell response, despite a lack of class switching in these mice. Through RNA-Seq approaches, the authors identify few differences between the lungs of WT and Igm-deficient mice, but see that two genes involved in actin regulation are greatly reduced in IgM-deficient mice. The authors target these genes by CRISPR-Cas9 in in vitro assays of smooth muscle cells to show that these may regulate cell contraction. While the study is conceptually interesting, there are a number of limitations, which stop us from drawing meaningful conclusions.

Strengths:
Fig. 1. The authors clearly show that IgMKO mice have striking reduced AHR in the HDM model, despite the presence of a good cellular B cell response.

Weaknesses:
Fig. 2.
The authors characterize the cd4 t cell response to HDM in IGMKO mice.
They have restimulated medLN cells with antiCD3 for 5 days to look for IL-4 and IL-13, and find no discernible difference between WT and KO mice. The absence of PBS-treated WT and KO mice in this analysis means it is unclear if HDM-challenged mice are showing IL-4 or IL-13 levels above that seen at baseline in this assay. The choice of 5 days is strange, given that the response the authors want to see is in already primed cells. A 1-2 day assay would have been better. It is concerning that the authors state that HDM restimulation did not induce cytokine production from medLN cells, since countless studies have shown that restimulation of medLN would induce IL-13, IL-5 and IL-10 production from medLN. This indicates that the sensitization and challenge model used by the authors is not working as it should. The IL-13 staining shown in panel c is also not definitive. One should be able to optimize their assays to achieve a better level of staining, to my mind.

In d-f, the authors perform a serum transfer, but they only do this once. The half life of IgM is quite short. The authors should perform multiple naïve serum transfers to see if this is enough to induce FULL AHR.

The presence of negative values of total IgE in panel F would indicate some errors in calculation of serum IgE concentrations.

Overall, it is hard to be convinced that IgM-deficiency does not lead to a reduction in Th2 inflammation, since the assays appear suboptimal.

Fig. 3. Gene expression differences between WT and KO mice in PBS and HDM challenged settings are shown. PCA analysis does not show clear differences between all four groups, but genes are certainly up and downregulated, in particular when comparing PBS to HDM challenged mice. In both PBS and HDM challenged settings, three genes stand out as being upregulated in WT v KO mice. these are Baiap2l1, erdr1 and Chil1.

Fig. 4. The authors attempt to quantify BAIAP2L1 in mouse lungs. It is difficult to know if the antibody used really detects the correct protein. A BAIAP2L1-KO is not used as a control for staining, and I am not sure if competitive assays for BAIAP2L1 can be set up. The flow data is not convincing. The immunohistochemistry shows BAIAP2L1 (in red) in many, many cells, essentially throughout the section. There is also no discernible difference between WT and KO mice, which one might have expected based on the RNA-Seq data. So, from my perspective, it is hard to say if/where this protein is located, and whether there truly exists a difference in expression between wt and ko mice.

Fig. 5 and 6. The authors use a single cell contractility assay to measure whether BAIAP2L1 and ERDR1 impact on bronchial smooth muscle cell contractility. I am not familiar with the assay, but it looks like an interesting way of analysing contractility at the single cell level.
The authors state that targeting these two genes with Cas9gRNA reduces smooth muscle cell contractility, and the data presented for contractility supports this observation. However, the efficiency of Cas9-mediated deletion is very unclear. The authors present a PCR in supp fig 9c as evidence of gene deletion, but it is entirely unclear with what efficiency the gene has been deleted. One should use sequencing to confirm deletion. Moreover, if the antibody was truly working, one should be able to use the antibody used in Fig 4 to detect BAIAP2L1 levels in these cells. The authors do not appear to have tried this.

Other impressions:
The paper is lacking a link between the deficiency of IgM and the effects on smooth muscle cell contraction.
The levels of IL-13 and TNF in lavage of WT and IGMKO mice could be analysed.

Moreover, what is the impact of IgM itself on smooth muscle cells? In the Fig. 7 schematic, are the authors proposing a direct role for IgM on smooth muscle cells? Does IgM in cell culture media induce contraction of SMC? This could be tested and would be interesting, to my mind.

Reviewer #3 (Public Review):

Summary:
This paper by Sabelo et al. describes a new pathway by which lack of IgM in the mouse lowers bronchial hyperresponsiveness (BHR) in response to metacholine in several mouse models of allergic airway inflammation in Balb/c mice and C57/Bl6 mice. Strikingly, loss of IgM does not lead to less eosinophilic airway inflammation, Th2 cytokine production or mucus metaplasia, but to a selective loss of BHR. This occurs irrespective of the dose of allergen used. This was important to address since several prior models of HDM allergy have shown that the contribution of B cells to airway inflammation and BHR is dose dependent.

After a description of the phenotype, the authors try to elucidate the mechanisms. There is no loss of B cells in these mice. However, there is a lack of class switching to IgE and IgG1, with a concomitant increase in IgD. Restoring immunoglobulins with transfer of naïve serum in IgM deficient mice leads to restoration of allergen-specific IgE and IgG1 responses, which is not really explained in the paper how this might work. There is also no restoration of IgM responses, and concomitantly, the phenotype of reduced BHR still holds when serum is given, leading authors to conclude that the mechanism is IgE and IgG1 independent. Wild type B cell transfer also does not restore IgM responses, due to lack of engraftment of the B cells. Next authors do whole lung RNA sequencing and pinpoint reduced BAIAP2L1 mRNA as the culprit of the phenotype of IgM-/- mice. However, this cannot be validated fully on protein levels and immunohistology since differences between WT and IgM KO are not statistically significant, and B cell and IgM restoration are impossible. The histology and flow cytometry seems to suggest that expression is mainly found in alpha smooth muscle positive cells, which could still be smooth muscle cells or myofibroblasts. Next therefore, the authors move to CRISPR knock down of BAIAP2L1 in a human smooth muscle cell line, and show that loss leads to less contraction of these cells in vitro in a microscopic FLECS assay, in which smooth muscle cells bind to elastomeric contractible surfaces.

Strengths:
1. There is a strong reduction in BHR in IgM-deficient mice, without alterations in B cell number, disconnected from effects on eosinophilia or Th2 cytokine production
2. BAIAP2L1 has never been linked to asthma in mice or humans

Weaknesses:

1. While the observations of reduced BHR in IgM deficient mice are strong, there is insufficient mechanistic underpinning on how loss of IgM could lead to reduced expression of BAIAP2L1. Since it is impossible to restore IgM levels by either serum or B cell transfer and since protein levels of BAIAP2L1 are not significantly reduced, there is a lack of a causal relationship that this is the explanation for the lack of BHR in IgM-deficient mice. The reader is unclear if there is a fundamental (maybe developmental) difference in non-hematopoietic cells in these IgM-deficient mice (which might have accumulated another genetic mutation over the years). In this regard, it would be important to know if littermates were newly generated, or historically bred along with the KO line.
2. There is no mention of the potential role of complement in activation of AHR, which might be altered in IgM-deficient mice
3. What is the contribution of elevated IgD in the phenotype of the IgM-deficient mice. It has been described by this group that IgD levels are clearly elevated
4. How can transfer of naïve serum in class switching deficient IgM KO mice lead to restoration of allergen specific IgE and IgG1?
5. Alpha smooth muscle antigen is also expressed by myofibroblasts. This is insufficiently worked out. The histology mentions "expression in cells in close contact with smooth muscle". This needs more detail since it is a very vague term. Is it in smooth muscle or in myofibroblasts.
6. Have polymorphisms in BAIAP2L1 ever been linked to human asthma?
7. IgM deficient patients are at increased risk for asthma. This paper suggests the opposite. So the translational potential is unclear.