Please don’t dig me out. I am writing this out of my conscience as a
scientist. We are using, largely, the tax payers’ money to explore
mechanisms underlying diseases that we don’t have a cure, yet. We are doing
this in the hope that little bit knowledge can accumulate till one day we
can find that magic bullet. However, there are people out there, their
purpose is to use these money to achieve their personal fames, sometimes
international ones, totally disregarding our original noble cause. I am
doing this because I believe a wrong message has to be corrected if it is
wrong, but I found I am now in no place to do that, I am expose this here in
the hope that somebody with power, can do this for us.
I am working in the inflammasomes field, where adaptor molecule ASC plays a
pivotal role by bridging interactions between NLR family members and the
proinflammatory Caspase-1. However, that paradigm was changed recently. In a
paper published in the 10th issue of volume 12 of Nature Immunology,
Ippagunta et al. claimed that they have identified an additional and
unexpected mechanism by which ASC, a core component of inflammasomes, can
control adaptive immune responses via regulation of the expression of the
gene encoding Dock2. The authors first demonstrated that primed dendritic
cells from Asc deficient mice are defective for uptaking of particulate
antigens. In addition, Asc deficient mice showed lower number of
lymphocytes as well as myeloid cells in peripheral lymphoid organs. More or
less, the phenotype presented in Asc deficient mice recapitulated the ones
discovered in Dock2 deficient mice. Gene expression array analysis followed
by Western blotting did indicate that Dock2 protein expression was abrogated
in Asc deficient lymphoid and myeloid cells. The authors further provided
evidence that messenger RNA for Dock2 has a much-shortened life span than
its wild type counterpart. Finally complementation of Asc deficient cells
with Dock2 protein did have rescued immune cell functions. The authors
concluded that they have identified a critical and unexpected role of Asc in
regulating the motility of lymphocytes and professional antigen presenting
cells independently of inflammasomes.
While the data presented in this paper are largely not flawed, the
interpretation of these data are quite questionable since the authors did
not provide evidence regarding complementation of Asc deficiency by forced
expression of ectopic Asc protein within these cells to determine whether
ASC regulates Dock2 expression.
We have the same Asc deficient mouse strain as the one the authors used in
their publication, which was originally generated by Millennium
Biopharmaceuticals. Inc. We have established stable bone marrow derived
macrophage cell lines (BMDM) from the original mouse line when we just
imported the mouse line. After a SNP analysis, we were not satisfied with
the amount of C57BL/6 genetic background in the mouse line and decided to
further back-cross them to B6 mice for two more generations.
We set out to explore the mechanism how Asc regulate Dock2 expression. When
we look at the stable BMDM Asc deficient cell line, we find that Dock2
protein expression is indeed absent. We took these cell lines and transfect
them with Asc expressing retroviral vectors. Ectopic Asc expression was
confirmed by Western blot, and pro-IL-1 processing, a hallmark of
inflammasomes activity, was restored by re-expression of Asc in these cells.
However, Asc protein re-expression was not able to rescue Dock2 expression
in these cells. We reasoned that the immortalization process by which we
have generated the cell line might have caused some changes that prevent
Dock2 re-expression after Asc complementation. We established primary BMDM
cultures with bone marrow cells from mice in our current Asc -/- mouse
colony. We were planning to work on these primary cells with the Asc
complementation experiment when, to our surprise, we found that these
primary cells are sufficient for Dock2 expression in the absence of Asc. We
took additional mice from our Asc-/- colony and found every one of them are
Asc deficient but Dock2 sufficient.
While this finding is surprising, we believe what really happened is that
the Asc-/- mouse strain originally from Millennium was a compound mutant
strain with mutations in both Asc and Dock2 locus. The nature of the Dock2
mutation in these mice remains mysterious to us. Our BMDM stable cell line
was established from the original Millennium mouse line, as a result these
cells are also deficient for Dock2 expression. However, after backcrossing
to B6 mice for two generations, that mutant Dock2 allele was segregated from
Asc deficient allele in our current Asc-/- mouse strain since Dock2 and Asc
encoding genes are located on different chromosomes. This explains why
reconstitution of Asc-/- cell lines with Asc protein expression vector
cannot rescue Dock2 expression.
It was a genuine mistake on the authors’ side, since they did not provide
complementation experimental data. It is also a mistake on the reviewers’
since they probably did not ask for such data. However, I believe we have
solid scientific evidence that the phenomenon observed by Ippagunta, et al
was no more than an artificial effect in an accidentally generated compound
mutant mouse line, thus the whole message in that paper does not stand.
It is truly an unfortunate situation for the authors, however, it is more
important to inform the community that they have made a mistake instead of
covering it up and let the wrong message be there to misled other scientists
or even finding its way into textbooks for the next generation.
