In this talk, Dr. Aleksandra (Ola) Kolodziejczyk recounts her scientific journey from early single‑cell RNA‑seq experiments during her PhD to her post‑doctoral work at the Weizmann Institute investigating acute liver failure (ALF). Using mouse models of acetaminophen‑induced ALF, she performed single‑cell transcriptomics on resident liver cell types (stellate, endothelial, Kupffer cells) and infiltrating immune cells (monocytes, neutrophils). She identified a shared activation program of 77 genes across the resident cells, whose promoters are enriched for MYC binding sites. Pharmacological inhibition of the MYC‑MEK axis (MEK inhibitor) abolished CCL2 expression, prevented monocyte infiltration, reduced tissue damage, and generated a distinct cellular state absent in untreated disease. Neutrophil recruitment, driven by CXCL2, remained unaffected, highlighting pathway specificity. Parallel experiments in germ‑free and antibiotic‑treated mice showed attenuated MYC activation, implicating the microbiome. Genetic ablation of TLR adaptor proteins MyD88 and TRIF phenocopied MYC inhibition, linking microbial‑derived TLR signaling to MYC activation via MAP‑kinase signaling. Human relevance was demonstrated by immunohistochemistry showing strong nuclear MEK up‑regulation in liver samples from ALF patients and transplant recipients. The work underscores a common, microbiome‑modulated transcriptional program in liver resident cells that drives monocyte recruitment, and illustrates how single‑cell approaches can uncover therapeutic targets. Acknowledgments include collaborations with Sarah’s lab (computational training), Alina’s lab (post‑doc), Eran Elinav’s lab, and clinicians at Tel Aviv Hospital.
Chapters
So as Sarah said, my PhD was very eventful in general. So I joined in 2012, and one thing that I would like to show you before I'm going to the science is I looked through my pictures and I wanted just to kind of show how far we went from the early days, like 2012, '13, of the single-cell sequencing to now. You have like a little picture of my lab book from December 2012 when we were trying to sequence 5 cells. I don't know. I can hear somebody.
Jose, I think you need to mute.
Yeah, so I think this is quite amazing. And the pictures I also picked because you can see kind of happiness and excitement.
I think you need to mute. Ponpan, I think you need to mute yourself. If you mute yourself, then we can hear Ola more clearly. Ponpan, thanks.
That's awesome. Thank you. Sorry. So I think these two pictures I picked because, well, they were also very early days. And they show the excitement that kind of was around during that time. And I don't know, I had the feeling that we are doing something special. And it was really great because of that. That we had the chance to do it, which also was not like planned for my PhD because I was supposed to do something completely different, and then this just landed on my lap, and this made me feel very special, and I'm really grateful for that. And also, I think I chose Sarah's lab because when I was applying for the PhD and I was looking for a lab where I could learn computational side of things. And for Sarah, that was obvious, and that I can do it, that it's not a problem. And she really believed in me, and that was really also very helpful, and, and I'm also very grateful for that. And what I would like to share, show you today, is I moved on to the Weizmann Institute to Alina's lab for my postdoc. Where I'm looking into the interactions between host and microbiome. In general, I'm interested in how external things, so environment and microbiome, it's a surface between human and environment, how they influence what happens in the human body. To look into that, we decided to study acute liver failure, which is a really horrible disease that is in humans most often a result of overdose of paracetamol. And then we did single-cell in mouse model of that. And what you can already see is that we observed a lot of differences between cell types. And when we quantified a little bit more, you can see that acute liver failure causes infiltration of cell types of monocytes and neutrophils to the liver. And activation of other cell types, stellate cells, endothelial cells, and Kupffer cells, which are resident cells of the liver. These activation signatures that happen seem to be quite similar when I looked at them. Then I found that there is a huge overlap between genes that become upregulated in all of these populations when they become activated. There's as many as 77 genes that are not expressed or lowly expressed in the quiescent versions,, and then they become activated and expressed in the activated versions of these cells in the liver disease. And the fact that these are similar suggests that there is a common regulatory mechanism in all of these cell types that drives that, and likely also a common kind of external signal that drives that in all of these cell types. And to identify what could that be? I looked at the promoters of these 77 genes, and I found that in the promoter there's a highly enriched binding site for Myc transcription factor in these 77 genes. So, to see if this is indeed a regulator, I used Myc inhibitor. And in mice, we performed this disease model using using MEK inhibitor to see if we can stop this, this response. And the readout that we use is infiltration of monocytes, because monocytes infiltrate into the liver through CCL2 chemokine. This chemokine is one of the 77 genes. So the logic was that if we stop MEK transcription factor, there will be no CCL2, and that will lead to no infiltration of monocytes. Indeed, what you can see here, if we give MEK inhibitor, there's no infiltration of monocytes. But on top of that, there's also less tissue damage, which we can see in histology, but also when we look at the liver enzymes in the blood, which is a common measure of liver damage, basically. And when we did single-cell sequencing in the presence of MEK inhibitor, we find in two different models that there is new population arising, which is different than the activated population in absence of MEK inhibitor, but also is different than the quiescent population that is in healthy animals. And this happens in all of the populations that are resident, so stellate cells and odontelial cells. For cells. And when we look at the expression of the signature, it appears that these genes, again, they are not expressed in the quiescent healthy mice, then become activated in the disease. But if we give MEK inhibitor, they are not there anymore, proving that indeed MEK is a transcriptional regulator for most of these genes in the signature, and especially for the CCL2, which is a driver of the infiltration of monocytes. Monocytes. And in the single-cell data, we can also see this abrogation of the monocyte infiltration because of CCL2 that's only expressed in activated cells but not in the presence of MYC inhibitor. But on the other hand, neutrophils that are also infiltrating in the disease are not affected by the MYC inhibitor. And this is because they are infiltrating through CXCL2, which is not affected by inhibition of MEK. So we can see this kind of specific effects of MEK inhibition. And it's not a kind of global effect. So neutrophils are not affected. And when we go to the microbiome and we, I performed the single-cell experiments also in mice that are germ-free. So mice that don't have microbiome at all, they are basically sterile throughout their life. And I inflict this acute liver failure in these mice, we can see that there is, first of all, less of serum liver enzymes, which are the measure of liver failure. Also, there is a higher expression of the 77 gene signatures of the mix signature in mice that are SPF, so normal colonized mice compared to mice which are germ-free. There's the same pattern in antibiotic-treated mice that also are devoid of microbiome. Heavily depleted microbiome. And this led me to the hypothesis that microbiome acts on the MYC through TLR signaling. And this would be in a way that damage-associated molecular patterns that are originating from damaged hepatocytes in disease normally would signal through TLR to MYC. And they would be— the signal would be added to the signal from pathogen-associated molecular patterns from microbiome. And that's why in mice that are devoid of microbiome, the MYC activation is a bit smaller than in mice that have microbiome. And TLR signaling is here the best candidate. And to check if indeed this signaling plays a role in this activation, we used mice that are knockout for MyD88-TRIF. And this is because there are many different TLRs. It's difficult to knock all of them out, but they have only these two different adaptor proteins, MyD88 or TRIF. It's easier to knock out these two adaptors. Then when I perform the acute liver failure model in these mice, I see that these cells, the activated cells from the liver, they look the same, they basically phenocopy what we see in MYC inhibition. So, lack of TLR signaling and MYC inhibition looks the same. So, there are the same populations in stellate cells and in endothelial cells. In Kupffer cells, it looks different because already at the steady state, the Kupffer cells look different, probably their development is affected by lack of the TLR signaling. And when we look at the expression of the 77 gene signatures, the MYC signature, also in MyD88-trif knockout mice in presence of APAP. So in acute liver failure, there's no upregulation in neither of the populations that are raised then. And also similarly to MYC inhibition, there is no infiltration of monocytes, but there is infiltration of neutrophils, shows that again, this is kind of specific to part of the phenotype. And after we established this, we wanted to see also if there is any effect in humans. And in mice, we observed not only these differences on the RNA level, but also we observed increase in the MEK protein levels with Western blotting. And in humans, we perform immunohistochemistry for MEK protein, and there's a huge upregulation of MEK in the nuclei of patients who have acute liver failure or who had acute liver failure and then they had liver transplants. And this work was done in collaboration with Dr. Shlomay from Tel Aviv Hospital. Yeah, so to sum up, I showed you that there's a common microregulated activation in stellate cells, endothelial cells, and Kupffer cells, and that it is regulated by the microbiome and it affects infiltration of monocytes. And also, I didn't show you, but we also look into how the TLR and MYC are connected, and they are connected through MAP kinase pathway. So we established that using different inhibitors also in mouse model. And in the end, I would like to thank the lab I'm in now, the Eran Elinav lab, And yes, that's all.
Yeah, fantastic talk, Ola, and thank you for the memories. It was a good time. I was wondering, what is the— like, is the signaling for the ingress signal for the monocytes and neutrophils via the Is that directly via the stellate cells?
So, they— so, stellate cells, it's all these actually cells express the chemokines that the whole 3, XCL2, it's kind of like a global—
And is that repressed? I mean, is the chemokine expression repressed through MYC pathway then?
So, yes. So, if I inhibit them, they are not there anymore. Okay. Okay.
