Summary of the CMS Seminar Club presentation on Friday, September 30, 2022.
Title: Mucosal tissue barrier functions — from early life development to immunity to infection
Speaker: Dr. Menno J. Oudhoff, Associate Professor, Department of Health Sciences, Faculty of Science, Carleton University, Canada.
On Friday, September 30, Dr. Oudhoff gave a presentation at Fujita Health University. He showed us how different types of immune responses change the intestinal epithelium and vice versa. The intestinal epithelium is not just a passive bystander or victim but actively changes its cellular components to meet immune challenges.
Recording: For members of Fujita University, a recording of the meeting (without the discussion part) will be available at our Manabi system. Unfortunately, we cannot open the recording for a wider audience.
There were 28 participants who enjoyed the meeting. Dr. Oudhoff gave a general introduction about the immune system and mucosal barriers and then focused on experiments by his group. They found that different immune responses (different immune “polarizations”) lead to changes in the epithelial cellular constituents of the intestinal epithelial layer, which in turn modify the immune responses. Such findings are very important because several intestinal diseases concern a chronically activated and polarized immune system (e.g., Crohn disease). The results shown by Dr. Oudhoff were clear and convincing and he explained them well and in a calm voice (it is truly worthwhile to watch the recording). Therefore, several participants with an immunology background told me afterward that they had highly appreciated the presentation.
For instance, Dr. Akihiko Nishikimi wrote: “I was impressed with his beautiful data. It also helped me to comprehensively understand gut immunity and epithelial cell function and differentiation.”
On the other hand, the non-immunologists with whom I spoke expressed that they could not understand large parts of the story. This may be because of the difficulty for non-immunologists to become sufficiently familiar during a single lecture with the terms “type 1,” “type 2,” and “type 3” immunology for understanding experiments done within this context. Hopefully, the summary of Dr. Oudhoff’s presentation below can help.
The contents of the presentation
This chapter is divided, as was the presentation by Dr. Oudhoff, in a general introduction about mucosal barriers, followed by descriptions of his own work. Not all the contents of his presentation are summarized here.
General introduction
Mucosal tissues are common initial battlegrounds for infection. They include the gastrointestinal tract (mouth-intestine), respiratory tract (trachea-lung), and reproductive tract. All these mucosal tissues have in common that they consist of an epithelial layer, associated microbiota (bacteria and other microorganisms), and a local repertoire of immune cells.
Dr. Oudhoff’s presentation focused on the gastrointestinal tract (Fig. 1), which in humans has ⁓90 m2 surface area. The intestine is ⁓6 m long and is home to ⁓100 trillion microbes. Especially in the colon, the number of microbes is very high, and they are kind of being “farmed” to help us with food digestion. Throughout our lives, we process about 35000 kg food, and we need to be immune-tolerant toward food as well as commensal microbes.
As we already learned from the presentation by Prof. Hans Clevers in February this year, the epithelia of both the small intestine and colon have “crypts” (indentations) in which at the bottom stem cells reside together with Paneth cells. However, only the small intestine has “villi,” which are protrusions into the intestinal lumen to increase the epithelium surface.
Intestinal epithelia keep rapidly generating new cells, which differentiate into different cell types:
• Stem cells, generate all intestinal epithelial cells
• Paneth cells, important for crypt organization and secretion of anti-microbial peptides
• Goblet cells, important for mucus production
• Enterocytes, important for food uptake
• Tuft cells, cells with brush-like microvilli that are important for type 2 (anti-parasite) immunity by secreting interleukin 25 (IL-25)
• Enteroendocrine cells, specialized cells with an endocrine function
Dr. Oudhoff explained to us that in the embryo there are no crypts and that the crypts, microbiome, and cell diversity increase from the day of birth to about three weeks after in mice which corresponds to about one year in humans (Fig. 2). This development coincides with the change from milk to solid food.
The intestinal epithelial stem cells divide unusually fast, and the intestinal lining is renewed about every five days. This helps to rapidly adapt the epithelial layer when challenged, and to rapidly return to normal after the challenge has gone. For example, Dr. Oudhoff showed that in the small intestine in the mouse, eight days after infection with a helminth worm N. brasiliensis, the epithelium underwent a massive change with an increase in number and activation of goblet cells (UEA-1 and RELM-β are markers) so that a lot of mucus could be produced to expel the helminths; then, seven days after the infection stopped, the epithelium was back to its pristine state again (Fig. 3).
For properly understanding Dr. Oudhoff’s presentation, the listener/reader should have some concept of the major possible polarizations of immune responses. Basically, the immune system has a large array of cell types and molecules that it can choose from to upregulate or downregulate an immune response. Generally, it does not want to activate them all at the same time and tends to activate them in clusters that downregulate other clusters. While the biological truth is more complex and far from well understood, three of the major immune “polarizations” that can be distinguished are (with highlighting cells and cytokines important in Dr. Oudhoff’s story):
Type 1 immunity. Function: Killing of infected cells and cancer cells. Activated cell types: Natural killer (NK) cells, CD8+ T cells, Th1 CD4+ T cells, ILC1 cells, IgG2a B cells. Marker cytokines: interferon-γ (IFNγ), IL-12
Type 2 immunity. Function: Killing of parasites (also involved in allergies). Activated cell types: Eosinophils, Basophils, Mast cells, Th2 CD4+ T cells, ILC2 cells, IgA B cells, IgE B cells. Marker cytokines: IL-4, IL-5, IL-9, IL-13.
Type 3 immunity. Function: Killing of extracellular bacteria and fungi. Activated cell types: Neutrophils, Th17 CD4+ T cells, ILC3 cells. Marker cytokines: IL-17, IL-21, IL-22.
The experiments performed by Dr. Oudhoff and co-workers
Essentially, the major two questions addressed by Dr. Oudhoff”s group in the experiments explained to us were: (1) How does the epithelium change from pristine to “immune-activated,” and (2) how does it return to pristine again (see Fig. 3).
A large part of the experiments he described were recently published in Science Immunology (Lindholm et al. 2022). While the basic setup of Dr. Oudhoff’s experiments was surprisingly elegant and simple, it was quite new and combined with the elucidation of regulatory details and confirmation in vivo, plus Dr. Oudhoff’s keen instinct for understanding the functional relevance of processes, lead to a publication in this top journal. I appreciated this even more after carefully listening to the recording.
Intestinal organoids change upon different immune stimulations
Dr. Oudhoff importantly used organoids from the small intestine (Fig. 4) for his experiments. As you may recall from Prof. Clevers’ beautiful presentation in February this year, these organoids are small sacks in which all forms of epithelial cell differentiation take place as well as crypt formation. An advantage of the system is that epithelial processes can be studied in isolation because there are no other cell types.
Dr. Oudhoff incubated these organoids with the cytokines IFNγ (a stimulator of type 1 immunity), IL-13 (a stimulator of type 2 immunity), or IL-22 (a stimulator of type 3 immunity), and found morphological (Fig. 1B in Lindholm et al. 2022) as well as gene expression (Fig. 1F in Lindholm et al. 2022) differences. These three cytokines all are known to stimulate intestinal epithelial cells (Onyiah and Colgan 2016). As an example of differential regulation, the high upregulation of Bmp2 gene was found to be unique for IL-13 (Fig. 3C in Lindholm et al. 2022.
What happens to the different epithelial cell types upon different immune stimulations?
Each cell type has its own characteristic gene expression profile, and by gene set enrichment analysis (GSEA) it can be estimated which cells are particularly stimulated by the different cytokines. In this way, Dr. Oudhoff found, using his organoid experiments, that IFNγ stimulated enterocytes, IL-13 stimulated tuft cells, goblet cells, and Paneth cells, and IL-22 stimulated goblet cells (Figs. 2A and 2B in Lindholm et al. 2022). A large part of Dr. Oudhoff’s presentation was dedicated to goblet cells.
Although both IL-13 and IL-22 stimulated goblet cells, the sets of genes were different (Fig. 2B in Lindholm et al. 2022) and in the case of IL-13 were predominantly related to goblet cell proliferation (differentiation from precursor cells) and in the case of IL-22 to goblet cell immune activation; this was confirmed by fluorescent labeling of stimulated organoids for the lineage marker mucin by IL-13 and the immune-activation marker RELM-β by IL-22 (Figs. 2D and 2E in Lindholm et al. 2022).
Dr. Oudhoff also confirmed the relevance of this finding in vivo. Namely, infection of the mouse small intestine with the helminth (a worm parasite) Nippostrongyius brasiliensis— which induces type 2 immunity (thus involves IL-13)—a large increase in goblet cells and tuft cells was observed (Fig. 5 top), and when infected with the bacterium Citrobacter rodentium—and inducer of type 3 immunity—the goblet cells were activated to express more RELM-β (Fig. 5 bottom), which is a molecule with bactericidal properties (Propheter et al. 2017). The intestinal gene sets that were stimulated with these two different pathogens also corresponded well with those stimulated in the organoids by IL-13 and IL-22 (Fig. 1G in Lindholm et al. 2022).
Dr. Oudhoff speculated that for fighting helminths a lot of mucus is needed, so that the number of goblet cells should increase, whereas for fighting bacteria the response may have to be so rapid that there is no time to first generate new goblet cells (Fig. 6).
He also discovered a mechanism by which IL-13 does enhance the number of goblet cells. Namely, it induces the expression of ATOH1 (Fig. 7) which is a transcription factor expressed in cells in the crypts of intestinal epithelium (or organoid) that helps decide the faith of newly generated cells through a NOTCH signaling pathway. ATOH1+ cells become goblet cells or Paneth cells (Lo et al. 2017). In contrast, IL-22 or IFNγ do not induce ATOH1 expression (Fig. 2H in Lindholm et al. 2022).
The negative feedback loop that helps prevent type 2 immunity from spiraling out of control
The next part of Dr. Oudhoff’s seminar dealt with processes that help the intestinal epithelial to return to normal after an infection (the last step in Fig. 3). He was particularly interested in finding a negative feedback loop for type 2 immune activation of tuft cells. Namely, type 2 immunity increases the number of tuft cells and they are known to express IL-25, which is a type 2 immunity cytokine that stimulates tissue-resident ILC2 (type 2 innate lymphoid cells) immune cells to release IL-13. That would create a positive feedback loop (Fig. 8), and Dr. Oudhoff tried to find the brake of the system. He then turned his eye to the cell differentiation systems in the intestinal epithelium, one of which is the BMP (bone morphogenetic protein) system. He found the BMP system to be stimulated by IL-13 (Fig. 4D in Lindholm et al. 2022), including the enhanced Bmp2 expression (Fig. 6), and that IL-13-mediated enhancement of BMP system genes could be blocked by the inhibitor DMH1 (dorsomorphin homolog 1, a selective inhibitor of activin receptor-like kinase 2 [ALK2] which functions in BMP signaling) (Fig. 4D in Lindholm et al. 2022).
As evidence that BMP signaling inhibited tuft cell differentiation, Dr. Oudhoff found that DMH1, as an inhibitor of BMP signaling, increased the number of tuft cells both in organoids (Fig. 4F in Lindholm et al. 2022) and in vivo (Fig. 9). Hence, BMP signaling participates in a negative feedback loop that helps prevent activated tuft cells from continuously generating more tuft cells and helps the intestinal epithelium to return to normal after the pathogen challenge is over (Fig. 10).
It is of note that the IL-13-induced BMP signaling specifically involves the tuft cells (Fig. 4F in Lindholm et al. 2022); Dr. Oudhoff found that these cells express BMP2 and proposes that this molecule plays an important role in the negative feedback loop (Lindholm et al. 2022).
More in general, Dr. Oudhoff proposes that immune factors can kind of “hijack” parts of the normal epithelial cell differentiation system—like the BMP system in the case of type 2 immunity—to generate an epithelium suitable for the immune challenge at hand.
Epigenetic modifier LSD1 mediates the early life switch in intestinal epithelial maturation
The last part of Dr. Oudhoff’s seminar discussed the issue explained in Fig. 2, namely that it takes a while after birth before an adult type of intestinal epithelium is established. I will only summarize this very short and not show pictures. The data have been published in Zwiggelaar et al. 2020.
Dr. Oudhoff found that genes for intestinal Paneth cells, which are necessary for adult-type crypt formation, only started to be expressed at three weeks of age in mice. He assumed that this should be accompanied by stable epigenetic changes, and tested a number of epigenetic modifier inhibitors in organoids. Starting with the results from those experiments, he found that lysine-specific demethylase 1A (Kdm1a/Lsd1) is necessary for Paneth cell differentiation. He established knockout mice that lacked LSD1 expression in epithelial cells and their intestinal epithelium appeared to have no Paneth cells and to carry neonatal characteristics (for such features see Fig. 2) into adulthood (for details see Zwiggelaar et al. 2020).
So, his group is not only contributing to the elucidation of cell type differentiation in the intestine, but also of how the intestine changes from neonatal to adult.
