Tag Archives: NOD2

New Insights about NOD2 and Th17 Differentiation

microRNA Mir210
This little piece of RNA has the power to influence Th17 differentiation.
This week on TIBDI: NOD2 and IFNγ work together to recruit cells to the small intestine, and a microRNA offers an interesting way to control Th17 differentiation.

NOD2 Behind Intestinal T Cell Recruitment

One of the most important receptors involved with Crohn’s disease (CD) is NOD2, a pattern recognition receptor that recognizes bacterial cell walls. Dr. Xingxin Wu of the Yale University School of Medicine investigated its involvement in an acute intestinal disease model induced by systemic anti-CD3. His results provide unique insight into infiltration dynamics of the characteristic CD8+ T cells found in the small intestine of this model. He discovered that NOD2 stimulation was needed for optimal infiltration. Without these signals, chemokines, specifically CXCR3-ligands, were not secreted by macrophages, dendritic cells and stromal cells. This prevented CD8+ T cells from leaving the circulation and entering the intestinal lamina propria. Moreover, the loss of CD8+ T cells in the small intestine led to reduced IFNγ, which also plays a role in stimulating immune cell chemotaxis.

Unexpected MicroRNA Control of Th17

During low oxygen conditions, immune cells upregulate transcription factors that turn on genes that help them cope with the hypoxia. One of these transcription factors, HIF-1α, also contributes to the differentiation of Th17 cells, which are important in the pathogenesis of inflammatory bowel disease (IBD). In an extremely interesting Nature Immunology publication, Dr. Haopeng Wang of the University of California in San Francisco described how the microRNA Mir210 inhibited HIF-1α expression and Th17 differentiation. MicroRNAs are small RNAs that prevent gene expression. By controlling the abundance of Mir210, he was also able to influence the numbers of Th17 T cells differentiated in vitro. Using the T cell transfer model of colitis with genetically manipulated T cells, which lacked Mir210 expression, he found that Mir210-deficient T cells caused increased numbers of Th17 and worsened symptoms. The authors suggest that drugs that function similarly to Mir210 could be interesting therapeutics.

References

  • Wang, H., Flach, H., Onizawa, M., Wei, L., McManus, M. T., & Weiss, A. (2014). Negative regulation of Hif1a expression and T. Nature Immunology, 1–10. doi:10.1038/ni.2846
  • Wu, X., Lahiri, A., Haines, G. K., Flavell, R. A., & Abraham, C. (2014). NOD2 Regulates CXCR3-Dependent CD8+ T Cell Accumulation in Intestinal Tissues with Acute Injury. The Journal of Immunology. doi:10.4049/jimmunol.1302436

X-linked and Autophagy Genes Support Crohn’s Disease Development

Caspase 3 subunits
Caspase-3 is the missing link between an autophagy gene mutation and heightened inflammation.
This week on TIBDI: Impressive results in Nature show how gene mutations cause reduced autophagy, and mutations in the XIAP gene lead to early-onset CD in male patients.

From an Autophagy Gene to Crohn’s Disease

Genome wide association scans confirmed the importance of ATG16L1 mutations in Crohn’s disease (CD), especially a variant consisting of an Alanine to Threonine exchange at the 300th amino acid. Despite an abundance of literature, the precise mechanism linking the mutation to reduced autophagy was unknown. Dr. Aditya Murthy from Genentech, Inc. has now found an answer. The mutation is located in a cleavage site for the enzyme caspase-3, and it makes the protein more susceptible to cleavage. Caspase-3 is well known for its role in initiating apoptosis during cellular trauma, for instance during metabolic stress or intestinal infection. Without proper autophagy, macrophages are unable to neither regulate their energy consumption nor properly eliminate pathogens, and have an heightened inflammatory response.

XIAP Mutations in Early Onset CD

Variants in the gene encoding for the X-linked inhibitor of apoptosis protein (XIAP) can sometimes lead to intestinal inflammation. XIAP is involved with a multitude of processes including NOD signaling, apoptosis and NKT cell development. To investigate XIAP’s possible role in CD, Dr. Yvonne Zeissig and her colleagues at the University Medical Center Schleswig-Holstein in Germany, looked at CD patient samples to find if there were clear associations between XIAP and immune cell function. She found that approximately 4% of male early-onset CD patients had unique mutations in their XIAP genes. Experiments with patient primary cells revealed that loss of XIAP function caused defects in NOD1/2 signaling.

References

Insight: What is Autophagy?

Mycobacterium tuberculosis Ziehl-Neelsen stain 02
Autophagy is also responsible for the destruction of Mycobacterium tuberculosis.
After genome wide association scans discovered that autophagy genes were involved in Crohn’s disease (CD), autophagy suddenly became an interesting research topic. Autophagy, however, actually is a somewhat general term that encompasses a wide variety of processes. Below is a short overview of the basics and what you need know to understand its involvement in CD.

Autophagy, which means “self-eating” in Greek, refers to when cells digest cytoplasmic contents in lysosomes. Those of you who are familiar with lysosomes will probably realize that autophagy must be occurring in a wide variety of situations ranging from simple protein recycling to the destruction of internal pathogens. This is correct, and this, most basic, form of autophagy is referred to as macroautophagy and requires the specialized autophagy-related proteins (ATGs), which include the famous, CD-related ATG16L1.

Simple Autophagy:

During the first steps of autophagy, ATGs and other proteins drive the formation of the initial structure called the phagophore. The phagophore looks like a crescent moon and is derived from internal membranes from various sources (mainly the endoplasmic reticulum). Key proteins to remember that drive this process are Beclin 1, the kinase ULK1, LC3 proteins and γ-aminobutyric acid receptor-associated proteins (GABARAPs). The phagophore scoops up cytoplasm and closes forming a completed autophagosome. Degradation happens when the autophagosome fuses to a lysosome.

But what signals initiate autophagy? The signals are diverse and often related to the detection of danger. They can include loss of nutrients (a sign of infection), signaling through internal pattern recognition receptors (NOD-like receptors, TLR4), danger signals (HMGB1), pro-inflammatory cytokines (IL-1β and IFNγ), reactive oxygen species and even signals via co-stimulatory molecules (CD40). Important downstream molecules that need to be activated include Beclin 1 and TRAF6.

Autophagy and the Immune Response

Autophagy has the potential to modulate immune reactions at multiple levels besides simply providing a means for the destruction of invading internal pathogens like mycobacteria and viruses. It also controls the clean up of organelles and modulates the destruction of internal proteins. Autophagy plays such a basic role in the cells that numerous changes arise when it becomes deregulated. In particular, various immune consequences could arise like changes in protein presentation or even the increase of important microRNAs. Autophagy machinery is also involved in the secretion of stored cytokines and other mediators.

Autophagy and Crohn’s Disease

Individuals with mutations in NOD2 and ATG16L1 have a much greater chance to develop CD. One reason appears to be that these patients have a decreased ability to induce autophagy in response from signals from NOD2. NOD2, besides being a receptor for bacterial muramyl peptides in the cytoplasm, also has the job of recruiting ATG16L1 and allowing autophagy protein complexes to be initiated at the plasma membrane near bacterial entry. The most common CD-associated mutation truncates NOD2 so that it is unable to localize ATG16L1 to the membrane. This would make cells more susceptible to mycobacterial infections.

There is an overlap of susceptibility between mycobacterial infections and inflammatory bowel disease, and there was a time when it was widely believed that CD could be related to a mycobacterial infection similarly to Johne’s disease in cows. Seeing that the mutations in NOD2 and ATG16L1 could be detrimental to the proper elimination of mycobacteria could be another reason to resurrect the hypothesis.

However, there is another interesting option as well. These same mutations also impact the ability of Paneth cells to secrete anti-microbial peptides in response to intestinal bacteria. This was also mentioned in last weeks post. It could also be that these mutations are also upsetting the relationship between the gut and the neutral microorganisms that inhabit them.

References

Controlling Intestinal Lymphocytes and Abnormal Paneth Cells

Complete intestinal metaplasia in a case of chronic gastritis, HE 3
Paneth cells, the red cells in the crypts, become abnormal in Crohn’s patients with NOD2 and ATG16L1 mutations.
This week, there are interesting insights about intestinal lymphocyte population maintenance with exciting functions for TGFβ and NOD2. Also a unique perspective uncovers relationships between Crohn’s disease mutations and Paneth cell function.

TGFβ-directed Memory Retention

Memory; it’s the characteristic of the adaptive immune system that allows it to react quickly and specifically. Resident memory CD8+ T cells (Trm) found in the intestines are a valuable subset of cytotoxic T cells that defends against intestinal viral pathogens. However, the signals that mediate this population were unclear. The recent article by Zhang and Bevan sheds some light on how this works. To do this, they created a transgenic mouse with TCRs specific for a model virus and lacking a transforming growth factor (TGF)-β receptor. With this unique tool, they were able to determine that TGFβ plays two important roles for Trm during inflammation. 1) It inhibits the migration of dividing CD8+ T cells from the secondary lymphoid organs during the beginning of inflammation, and 2) it helps retain the same cells in the intestines during the later stages of inflammation.

NOD2 Maintains Intraepithelial Lymphocytes

Despite the association between NOD2 and Crohn’s disease being known for more than ten years, how NOD2 functions in the gut is only now be unraveled. In a recent article in the Journal of Experimental Medicine, it was found that Nod2 deficient mice lack intraepithelial lymphocytes (IELs). IELs are mainly CD8+ T cells and γδ+ T cells and appear to have a protective role in inflammatory bowel diseases. The loss was mediated largely by deficiencies in proliferation and survival. Further investigation revealed that Nod2-microflora interactions were required along with Nod2 signaling on antigen presenting cells and interleukin-15 production to have optimal populations of IELs and protection from colitis.

Crohn’s Mutations and Paneth Cell Phenotypes

Genome wide association scans are an interesting way to learn more about diseases like Crohn’s disease. However, they provide an enormous amount of data. To gain a clearer view of the possible effects of associated mutations, VanDussen et al decided to narrow their scope and focus only on the function of Paneth cells. Paneth cells are the gatekeepers of the intestinal crypts and produce anti-microbial peptides to control bacteria infiltration. The researchers observed that high numbers of defect alleles for NOD2 and ATG16L1 led to high amounts of abnormal Paneth cells in Crohn’s disease patients. This was also associated with altered immune activation, changes in granuloma numbers and disease recurrence.

References

NOD2, Not Just Another Boring Receptor in Crohn’s Disease

NOD2 CARD15
NOD2 null mutations are associated with Crohn’s disease.
This week, the Crohn’s disease-associated receptor NOD2 gets some deserved attention. Researchers delve into the workings of the Nod2 knock-out mouse, while others look at the downstream mediator, Pellino3. Finally, a related protein, CARD9, shows a strong relationship with Th17 development.

NOD2 is one the most well known genes associated with Crohn’s disease (CD). Initial research regarding its role in immunology revealed that it’s an internal pattern-recognition receptor (PRR) that allows cells to detect invading bacteria and initiate pro-inflammatory cascades. This discovery led to some confusion as the null variation of the gene was associated with CD patients. Many asked the question, “how can the loss of something pro-inflammatory lead to more inflammation in the intestinal tract?” A Nod2-/- mouse was created, and, surprisingly, it did not develop spontaneous colitis, which stumped researchers even more. Although much research has been performed since the knock-out mouse was developed in 2005, there is still a lot to be learned.

Nod2-/- Mice Have Increased Intestinal Regulatory T Cells 

Amedola et al attempted to find out why Nod2-/- mice did not get spontaneous colitis. They found that the main reason appeared to an increase of regulatory T cells (Tregs) with latent transforming growth factor-β (TGFβ). The population of these cells seemed to be dependent on the intestinal flora and the increased intestinal permeability of the Nod2-/- mice. Their ability to modulate colitis susceptibility was confirmed with transfer experiments. In a nutshell, their results showed that, in actuality, Nod2-/- mice were actually more resistant to some intestinal insults than their wild type counterparts, including TNBS colitis and ethanol-induced colitis.

Pellino3, a Downstream Mediator of Nod2

Underneath it all, Nod2 is a receptor like any other that initiates a signaling pathway. Studies under the “cellular” hood are also extremely useful for learning about how this PRR functions. Yang et al has now revealed that one mediator, Pellino3, is particularly important to Nod2 signaling. Pellino3 was revealed to be an ubiquitin ligase and performed its job on RIP2. Ubiquitination of RIP2 is needed for the activation of NF-κB and MAPKs via Nod2 stimulation. Loss of Pellino3 reduced the ability of cells to produce cytokines after Nod2 stimulation. Interestingly, loss of Pellino3 was also noted in CD patients, suggesting a loss of NOD2 signaling in patients. Pellino3-/- mice were also more susceptible to TNBS and Citrobacter colitis than wild types, which combined well with the human data. However, some of you may note that this TNBS result is different than what one would expect after reading Amedola article mentioned above.

CARD9, Not Just Another CARD in the Pack

Another name for NOD2 is CARD15. CARD stands for caspase recruitment domain-containing protein. As some of you might know, caspases are enzymes needed to process large proteins into more active, small ones. A good example of this is caspase 1, which cleaves the interleukin-1β protein into its active form. Sokol et al looked at CARD9, which functions as an adaptor protein downstream of PRRs that sense fungi. Using a Card9-/- mouse, they found that Card9 was needed to induce pro-inflammatory cytokines during intestinal trauma caused by DSS-induced colitis. Contrary to what one might first think, this actually led to a more severe colitis and a slower recovery. In particular, these mice had defective Th17 responses and loss of innate lymphoid cells. This was further confirmed in the Th17-restricted Citrobacter-colitis model, where the mice were unable to shake the infection. The authors refreshingly suggest that the loss of effective Th17 is actually not helpful and may actually be the reason for increased intestinal inflammation.

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References:

Amendola, A., Butera, A., Sanchez, M., Strober, W., & Boirivant, M. (2013). Nod2 deficiency is associated with an increased mucosal immunoregulatory response to commensal microorganisms. Mucosal Immunology. doi:10.1038/mi.2013.58

Sokol, H., Conway, K. L., Zhang, M., Choi, M., Morin, B., Cao, Z., et al. (2013). Card9 Mediates Intestinal Epithelial Cell Restitution, T-Helper 17 Responses, and Control of Bacterial Infection in Mice. Gastroenterology. doi:10.1053/j.gastro.2013.05.047

Yang, S., Wang, B., Humphries, F., Jackson, R., Healy, M. E., Bergin, R., et al. (2013). Pellino3 ubiquitinates RIP2 and mediates Nod2-induced signaling and protective effects in colitis. Nature immunology, 14(9