Akkermansia Muciniphila induces chronic extramedullary hematopoiesis through cooperative IL-1R and TLR signals

  1. Yuxin Wang
  2. Tatsuya Morishima
  3. Maiko Sezaki
  4. Gaku Nakato
  5. Shinji Fukuda
  6. Yuhua Li
  7. Hitoshi Takizawa

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Abstract

Bacterial infections can activate and mobilize hematopoietic stem and progenitor cells (HSPCs) from the bone marrow (BM) to spleen, which is termed as extramedullary hematopoiesis (EMH). Recent studies suggest that commensal bacteria, particularly the microbiota, regulates not only the host immune system but also hematopoietic homeostasis. However, the impact of gut microbial species on hematopoietic pathology remains largely unknown. Here we found that systemic injection of Akkermansia muciniphila ( A. m .), a mucin-degrading bacterium abundantly existing in the human gut rapidly activates BM myelopoiesis, and induces a slow but long-lasting hepato-splenomegaly, characterized by the expansion and differentiation of functional HSPCs, which we termed chronic EMH. Genetic deletion of Toll-like receptor-2 and -4 (TLR2/4) partially diminished A. m .-induced chronic EMH, while additional pharmacological inhibition of the interleukin-1 receptor (IL-1R) completely alleviated splenomegaly and EMH. Our results demonstrate that cooperative IL-1R- and TLR-mediated innate immune signals regulate commensal bacteria-driven EMH, which might be relevant for certain autoimmune disorders.

Article Summary

The aim of our study is to understand how Akkermansia muciniphila (A . m . ) , one of the major mucin-degrading microbial species in the human gut activate the immune and hematopoietic systems in a mouse model. We found that a single injection of the A . m . membrane fraction can induce long-lasting hepatosplenomegaly with splenic EMH through cooperative IL-1R- and TLR-mediated innate immune signals.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary

    In this manuscript, Wang and colleagues demonstrate that a single systemic injection of a high dose of Akkermansia muciniphila (A.m.) lysate drives a rapid pancytopenia followed by prolonged anaemia and hepatosplenomegaly with late-onset extramedullary hematopoiesis (EMH). The latter, as well as the splenomegaly, were likely mediated through activation of pattern recognition receptors and IL-1R signalling pathways. This was demonstrated through the partial and full phenotype reversal in Tlr2;4-/- and MyD88;Trif-/- mice, respectively. Moreover, the phenotype was partially reversed following IL-1R antagonism. After performing multiplex protein assays and flow cytometry, the authors conclude that EMH, in this model, is mediated by IL-1a produced within the spleen by local monocytes and DC.

    Overall, the manuscript by Wang et al. is quite well presented, the experiments are mostly well controlled, the methods are well reported, and the data fit a clearly defined story with clinical relevance. Nevertheless, there are several major concerns that if addressed would greatly increase the strength of the authors conclusions.

    Major comments

    1. The "two wave" hypothesis of hematopoiesis - first in the bone marrow (BM) and then in the spleen - is interesting. However, although an early wave of BM hematopoiesis would make sense, under these circumstances, I don't think the data are strong enough to support this hypothesis as they stand. For example, although the frequency of LSK cells increase, the numbers of most LSK subsets decrease. Given the decrease in the absolute number of BM cells 1d after A.m. injection, isn't it possible that the LSK cells are only proportionally increased relative to the remaining Lin- cells? What happens to the absolute number of LSK cells following A.m. injection?

    Also, describing "two distinct waves of HSPC increase in the A.m.-injected spleen" (Fig 2 & S2 titles) and describing a "first wave" of HSPC expansion in the BM (lines 396, 399, 402 etc.) is misleading for the following reasons: (i) the data strongly support a single wave of increasing HSPC in the spleen, peaking at d14, and (ii) there is no evidence HSPC are increased in the BM until d56, although there does appear to be an early increase in MPP. The language should be changed accordingly.

    1. The flow cytometry panel is not comprehensive enough to fully characterize the mature hematopoietic cell populations to the levels that are claimed here. For example, it is a stretch to assume that all B220- CD3- CD11c- cells are DC (splenic NK cells, eosinophils, monocytes and red pulp macrophages, for example, can express CD11c, particularly following inflammatory insult), or that CD11b+ F4/80+ SSC-hi cells are eosinophils, especially when eosinophils should be F4/80-lo are not known to express Ly6C in the spleen (For reference, see Immgen). These gating issues may explain the conspicuous absence of macrophages (should be F4/80+CD11b+Ly6C- and would also have a higher SSC than monocytes) in the plots. The B cell gate will also contain PDC, which express B220 (but can be easily excluded using Ly6C and CD11c). With respect to assessing the mature leukocyte populations in the spleen, relabelling the gates (CD11c+ cells instead of DC, F4/80+ myeloid cells instead of eosinophils) would suffice, however, these issues become a problem when trying to identify which cell populations express IL-1a.

    Due to the limited antibody panel used here, there is not enough evidence to suggest that DC and monocytes are producing IL-1a. Moreover, the histograms showing the changes in expression of IL-1a on the "DC" and "Mo" are not very convincing. How does the IL-1a staining look on a dot plot? Is there good separation between positive and negative? These plots need to be included. What happens if you gate on the IL-1a+ cells first, then phenotype them?

    Macrophages and splenic stromal cells are also likely candidates for IL-1a production. To assess which cell types are the true source of IL-1a, the authors need to repeat these experiments (namely, injecting A.m. and assessing IL-1a expression by leukocytes (and ideally also mesenchymal cells)) at d1 and d14, using a more comprehensive panel. Consider adding MHCII, CD64, Siglec F and CD24 to help differentiate between DC, MF, eosinophils and monocytes. CD45+ vs CD45- could be used as a minimum to assess the expression of IL-1a on leukocytes vs. stroma.

    OPTIONAL: The mechanism could be better defined using bone marrow chimeras to assess the different contribution of TLR2/4 signalling and IL-1R signalling on the hematopoietic vs. mesenchymal cell compartments.

    1. From these experiments, it is difficult to fully rule out a contribution from the adaptive immune system to the splenomegaly phenotype due to the marked difference in the size of BALB/c and MSTRG spleens at steady state. The authors should show the differences in spleen weight and total cell number as a % increase from control. The no of HSPC should also be normalized per gram of tissue weight or represented as a fold change compared to the relevant control groups.
    2. When using fluorescent imaging to compare the abundance of HSPC and other cell populations in the spleen, the authors should provide absolute quantification from multiple FOV and multiple mice.
    3. Finally, although the experiments are adequately replicated, the stats are not always appropriate. For example, a t-test shouldn't be used when there are >2 groups, or for a time course. This needs to be amended.

    Minor comments

    • Line 82-83: I'm fairly certain monocytes and inflammatory Ly6Chi cells are the same thing.
    • Line 83-84: "IL-1a is crucial for sustaining inflammatory responses, recruiting myeloid cells to infected tissue and inducing hematopoietic stem and progenitor cell (HSPC) mobilization and expansion both in vitro and in vivo" - I don't believe IL-1a has been shown to be crucial for either, even if it has been shown to play a role. If I am mistaken, please reference with a manuscript showing relevant phenotypes using KO mice.
    • Line 214: "Thus, we decided to use 200ug of lysate for the rest of all experiments." - is this what was usen for Figures 1A-C? This is not mentioned anywhere.
    • Line 227: "containing both non-hematopoietic cells and immature HSPCs" Please reference Fig. 1H here. Otherwise, it is unclear how you identified the "HSPC and other cell types" in Fig. 1G.
    • Figure S2A is described in text before Supp 1I-O and Fig S1H is not referenced in text at all.
    • It would be interesting to include what happens to hepatomegaly in MSTRG, Tlr2;4-/- and MyD88;Trif-/- mice.
    • Please define WBM. Presumably whole bone marrow?
    • Notably, CCL2 is increased in spleen lysate, BM lysate and serum. Given is role in myeloid cell mobilization from the BM, I would expect its role in the phenotype described here to at least be discussed.
    • HSPC LT gate includes MPP1, and should be labelled as such.

    Significance

    General assessment: The manuscript provided by Wang et al. describes, for the first time, a prolonged anaemia and hepatosplenomegaly with late-onset extramedullary hematopoiesis following a single systemic injection of A.m. lysate. The EMH phenotype appears robust and the data implicating TLR-signalling and IL-1a production are compelling. The work has clinical relevance as it increases our understanding of the factors driving EMH.

    There are two key limitations that let this study down. Firstly, the lack of depth in the flow cytometry panel used for immunophenotyping means it is not at all clear which cell types are producing IL-1a. Secondly, the authors use an enormous dose of bacterial lysate that is well above physiological levels, even following a loss of barrier integrity (e.g., in patients with IBD). This makes me question the biological relevance of the study, particularly with respect to Akkermansia translocation.

    Advance: With some improvement, this study will advance the field, in general. Previous work has looked at EMH following LPS injection, or live E. coli infection, however; the authors are able to demonstrate a distinct Akkermansia-specific effect that differs to that of LPS, membrane components of a different gram-negative bacteria, B. theta. The advancements implicate IL-1a in the modulation of EMH, for the first time, providing some mechanistic insight into this phenomenon.

    Audience: This work will likely be of interest to basic researchers interested in EMH. It may also be of interest to clinical researchers of pathologies where EMH is a known complication, such as rheumatoid arthritis and cancer. The impact of the work will depend on whether or not EMH contributes to pathogenesis, or is an epiphenomenon. To my knowledge, this has not been fully established, although this is not my area of research.

    I am a basic researcher with expertise in immunology focused on host-microbe interactions, both within the intestine and at distal tissues. I have knowledge of BM hematopoiesis and the microbial factors that influence if although my knowledge on extramedullary hematopoiesis is limited.

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    Referee #2

    Evidence, reproducibility and clarity

    In this study, Wang et al reported single injection of Akkermansia muciniphila (A.m.) induces two waves of extramedullary hematopoiesis (EMH), and demonstrated the mechanism of "second wave" was contributed by IL-1α secretion in spleen DCs and Ly6C+ monocytes. It is an important work on understanding infection-induced EMH. However, several major concerns about claims in this manuscript need to be addressed.

    1. The authors demonstrated that A.m.-induced EMH were alleviated by knockout of Tlr2;4 or Myd88-Trif, or even IL-1R inhibition. EMH in the spleen is a mechanism by which the hematopoietic system responds to stresses. Therefore, whether inhibition of EMH by these ways can affect normal hematopoiesis in mice? Do mice have pancytopenia? Will the function of HSC in bone marrow be affected?
    2. In Fig.S1E, why did the WBC and PLT recover quickly after the first day, while the RBC took 14 days to recover? Are WBC and RBC regulated by two waves of EMH, respectively?
    3. The authors should show absolute numbers of each cell type, not just the percentage of immunophenotypically defined cells. For example, in Fig.1G, I, 2B.
    4. In transplantation assays, whole BM cells or splenocytes was use. However, the proportion of HSCs in BM and spleen were all changed post A.m injection, which could affect the outcome of chimerism rate after transplantation. Transplantations should be done on by transplanting sorted fresh immunophenotypic HSCs.
    5. The concentration of IFN-γ in both spleen and serum were increased continuously from D1 to 14. Would IFN-γ cause the second wave of EMH? Relevant assays for exclusion are necessary.

    Significance

    It is an important work on understanding infection-induced EMH.

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    Referee #1

    Evidence, reproducibility and clarity

    Wang et al. examined the mechanisms of splenic extramedullary hematopoiesis upon systemic injection of Akkermansia muciniphila in mice. They showed that components of this mucin-degrading bacterium mobilize bone marrow hematopoietic cells and induce splenomegaly by MYD88/TRIF-dependent innate immune signaling pathways. Activation of TLRs and release of interleukin 1-alpha from splenic cells were then responsible for the expansion and differentiation of functional hematopoietic progenitors in the spleen. Genetic deletion of TLR2 and 4 restrained splenomegaly, while, pharmacological inhibition of IL1 receptor abrogated splenomegaly and extramedullary hematopoiesis suggesting their cooperation in the observed phenotype. It has been widely accepted that splenomegaly arises as a consequence of inflammation and that TLRs are major drivers of this process in the context of bacterial or viral infections. Here, the novelty relies on the potential circuit with the IL1alpha-IL1R axis as an additional driver of splenic extramedullary hematopoiesis. Although the results summarized above indicate that both TLRs and IL1 individually participate to some extent in splenomegaly after Akkermansia muciniphila administration, they fail to demonstrate that they concertedly do so in the spleen. In fact, the blockade of IL1R has a more profound impact.

    Significance

    My concerns are the following:

    • The authors mentioned that a specific lipid from Akkermansia muciniphila is able to trigger a non-canonical TLR2-TLR1 heterodimer to release inflammatory cytokines and regulate human immune response. Why TLR1 was not considered in the experimental strategy?
    • IL1alpha is up-regulated in several splenic cells (in particular in macrophages, Fig. S4F). To demonstrate a critical involvement of dendritic cells or monocytes, depletion studies or conditional mice models should be evaluated. What about megakaryocytes? Why were excluded from the analyses?
    • Interleukin 1alpha KO mice model should be also evaluated.
    • The paper is very dense and not easy to read and follow. English editing is required.
  5. Version published to 10.1101/2022.01.03.474846v1 on bioRxiv