Interferons and the immune system: an introduction to their function

Interferons and the Immune System

Eleonora Musella Prof. Guido Poli Basic Mechanisms of Disease, GP8 13/10/2023 Interferons and introduction to immune system Today, we close the chapter on cytokines, while in the second part of the lesson, we begin formally to talk about the immune system.

Interferon Cytokines

The peculiarity of interferons is that they are Cytokines that modulate other cell types, the imamune response, and others, but mostly they exert an antiviral effect: by stimulating a cell with interferons, we can induce the cell to switch to an antiviral mode. Interferons are subdivided into three categories:

• Type Interferons: encode for (N-a and \FN-0
• Type 2 Interferons: encode for IFN-y (already mentioned in the context of pro- inflammatory cytokines, as it is one of the most potent pro-inflammatory cytokines, yet maintaining the peculiar properties of antiviral interferon)
• Other interferons: e.g., type 3 interferon.

History of Interferons

The term interferon dates back to 1920-1930, when investigators observed the phenomenon of "viral interference", which means that a certain tissue/cell was exposed to a certain virus, and this would limit the possibility to get a second infection. Actually, this led to many other discoveries (e.g., apoptosis, CRISPR-CAS9 system ... ) in cell biology, medicine etc. In 1957, Isaacs and Lindemann studied this phenomenon, by infecting the chicken embryo chorioallantoic membranes by flu, and they observed that the heat-inactivated influenza virus could prevent the infection by live influenza virus. The hypothesis was that there was some viral protein, some viral component that was released or expressed, which would prevent the infection. Actually, the phenomenon of the interferon was not caused by a viral protein, but it was caused by a cell response to a viral infection: it was a host factor or a host protein, that was released, and that would prevent another infection. It took another fifteen to twenty years to show that interferons and interferon-activated genes, that are activating the interferon response, are encoded by the mammalian genome, and by early 80s*, the genes for the interferons were cloned and defined, and it was understood that there were different kinds of interferons, such as Interferon type 2, which is IFN-Y. It was also understood, for example, that IFN-a is not actually a single molecule, but it consists of about a dozen molecules (similar, with slight differences), encoded by different genes, and other additional five or 6 pseudo-genes.

1Eleonora Musella Prof. Guido Poli Basic Mechanisms of Disease, GP8 13/10/2023 On the other hand, IFN-ß comes in two, B1and B2. Another breakthrough came roughly 20 years ago, with the discovery of a very specialized cell type, part of our immune repertoire, called plasmacytoid dendritic cell (pCD). The reason why this subset of dendritic cells was discovered so late (think about the fact that, for example, they already knew about myeloid dendritic cells, very important for triggering the adaptive immune response) is that these cells constitute a tiny fraction of peripheral blood cells. Immunology first looks at peripheral blood, because that is the easiest way to have access to immune cells, but considering their small number, scientists put less effort on analyzing them. The bottom line is that this was not a real minority, in fact, if we look into tissues such as the liver, or other organs, we find them in much more quantities, and actually, on a single cell basis, these cells can produce up to one thousand-fold more interferons than other cell types (macrophages, for example). These cells are called plasmacytoid for their morphological appearance similar to the one of dendritic cells. There is a different type of interferon, called IFN-y. Before the introduction of interleukin classification, a lot of molecules had very descriptive names; there was a molecule called MAF (macrophage activating factor), and when they cloned it, they found out that it was IFN-y, so we don't talk about MAF anymore. This has a really potent pro-inflammatory effect, and IFN-y is the benchmark cytokine for the so-called Th1 type of immune response, which means CD4+ T cells that produce B cytokines and others to activate CD8+ T cells into an anti-inflammatory mode. *Note: when you see these time scales, think about COVID: in less than a year we got the vaccine, and in the first three weeks we got the coding of the virus, while now we are talking about decades, from one observation to the other ...

2Eleonora Musella Prof. Guido Poli Basic Mechanisms of Disease, GP8 13/10/2023

Classic Interferons (IFN)

Type 1 IFNs Signaling

use IFNs, in order to induce the cell for anti-viral response, require a signal transduction system. The signal transduction system is the JAK/STAT system.

1. There is a receptor, (FNAR), made of two chains ((NAR) and (NAR2). This receptor, IFNAR (interferon alpha receptor), also binds to IFN-B) and all the other type IFNs. The scheme is the same for more cytokines: interferons are produced, they cluster the two chains of the (FN receptor
2. This clustering will recruit Jak, in this case this type of kinase is Jak1 and Tyk2 (tyrosine kinase 2).
3. The complex Jak1/Tyk2 will recruit &TAT1/STATS, which forms a heterodimes.
4. They will associate with another factor RF9.
5. This is a trimeric complex,(SGF3 complex (IFN stimulated gene factor 3 complex).
6. ISGF3 complex will migrate to the cell nucleus.
7. ISGF3 6inds o specific target sequences in the genes called "IFN-stimulated response elements (SREs)
8. The outcome is transcription activation of numerous IFN stimulated genes (ISGs). They are so numerous, that now there is a web base catalog of genes activated by IFNs, of ISGs, that is www.interferome.org You can explore genes, and how they are controlled by interferons. Just to give an example: SARS-Cov2 and SARS-Cov1 interact with the ACE2 receptor (angiotensin-converting enzyme 2 receptor), which is part of the control of blood pressure. ACE2 is an IFN stimulated gene, so controlled by interferons. To be stimulated by IFNs, receptors need to be displaced so that the IFN can bind and transducers the signal. Once the cell is activated through this pathway, there will be the activation of the classic IFN response.

Genes Activated by Interferon

We know now that there are thousands of genes activated by interferons, so it would be very complex to describe all of them, but there are master genes that are activating and defining the basic modality through which IFNs can lead to an antiviral response:

Type I IFN Type III IFN IFNAR1 IFN IEN IFNAR2 IFNLR1 IL1OR2 TYK2 JAK1 TYK2 JAK1 STAT STAT STAT P P STAT1 STAT2 STAT STAT P ISGF3 STATI O STAT2 IRES Types I and III STAT ISGs IRF9 P STAT1 ISRE main *

3Eleonora Musella Prof. Guido Poli Basic Mechanisms of Disease, GP8 13/10/2023 The first is Protein Kinase R (PKR), it will shut off translation, the total protein synthesis. This means that if we stimulate a cell with IFNs, the cell will shut off the translation of its own proteins, as well as viral proteins. The second one is 2',5'-Oligoadenylate Synthetase (OAS), which synthesizes this oligoadenylate product which works on ATP and this will activate a hydrolytic enzyme that will cleave RNA) called RNase ). RNAase L does the same thing that PKR does at the protein level, but for RNA: it degrades both viral and CellularRNAs) So. by shutting of protein translation, the IFN is shutting off cell function. In the evolutionary selection game, this kind of response is protective for the individual. Additionally, there are proteins that have a role in blocking dral replication, for example Mx1 and Mx2) biochemically, they are GTPases, and are very effective against lofluenza virus (but also other viruses, including HIV, Covid ... ). Again, there are hundreds of other interferon-stimulated genes (SG): e.g., in the Professor's lab, they are studying one specific gene, called TRIM22, which is very strongly induced by IFNs. Remember: the real signature of IFNs are the genes PKR, shutting off protein translation, OAS, and RNase L, that leads to the degradation of all RNAs. There are other numerous genes and gene products that, overall, turn the cell into an antiviral mode.

Other Type 1 IFNs

Over time, other IFNs belonging to the type 1 group were discovered:

• (FN-K is selectively produced by keratinocytes when the skin s iciured.
• (FN-8, is constitutively secreted in the female genital tract; this is a natural antiviral agent, that is regulated by estrogen (rather than by cytokines) The mechanism of action is the same; IFN stimulates the cell in the same way, but what is differential is the site of production: the skin for k and female genital tract for & (modulated by estrogen, already regulating menstrual cycle and pregnancy). In addition, (FNs also participate to the orchestra of the immune response, and indeed they also activate:
• 1 - A very important kinase, called p38 mitogen-activated protein kinase (MAP kinase), then PI3K and AKT, utilizing transcription factors.
• (p53, an oncosuppressor gene that promotes DNA repair and apoptosi's

4Eleonora Musella Prof. Guido Poli Basic Mechanisms of Disease, GP8 13/10/2023 It is not surprising that IFNs, when they were discovered, could be the ultimate cure for cancer. Actually, there are some cancer types that are cured by interferons, even if not as many as we would desire.

Type 2 IFN Signaling

Type I IFN Type III IFN Type II IFN Type 2 IFN Signaling (and differences with Type 1 IFN) Again, we have the exploitation of the (AK/STAT pathway, but with a different combination of JAK and STAT. 2 IFN-y type 2, has two Seceptor chaing (ENGR) and (ENGR2, that will cluster and will recruit JAK? and (AK2) The consequence is the activation of a Types I and III STAT > ISGs homodime) of STAT? (remember that in type 1 we STAT1 P IRF9 ISRE VI GAS had a heterodimer of STAT1 and STAT2). The STAT1 STATI D goto: homodimers, without needing to complex with additional factors in the cytoplasm, go straight to the nucleus and they bind to GAS sequences (gamma interferon activating sequence). They bind to different ONA target sequences, but not to ISRE (bound by IFN type 1). Remember that type 1 can also bind to GAS sequence, but type 2 cannot bind to ISRE, so there is some cross-function.

Discovery of CGAS and CGAMP

In the last 10 years, it was understood that there were additional components of the IFN response. We will see later that the first interferon secreted upon an Infection is (FN-B) not IFN-a. ATP + GTP CGAS CGAMP IRF3 STING NFKB It was seen that there is an additional modality to produce the first interferon (IFN-B). There is this enzyme called cGAS, which is triggered by cytoplasmic DNA> cGAS combines ATP and GTP and produces cGAMP (cyclic GMP- AMP). cGAMP activates a gene called STING ("Stimulator of IFN Genes"), that Induces the transcription factors (RF3 and NF-KB to secrete (FN-B) AVAY dsDNA CGAS STING CGAMP immune activation Type 1 interferon The peculiarity of this modality of activation is that ATP + GTP STING can act on cGAMP, but also it can diffuse T into neighboring cells, so it can expand the ENPP1 activation of the IFN response also to cells that cancer cell host cell have not yet been infected. So, not only the infected cells can amplify the antiviral response, but it is also possible to put the other neighboring cells into an antiviral mode. IFNGR1 IFNAR1 IFN IFN IFN X IFN ENAR2 IFNLR1 IL1OR2 IFNGR2 Plasma membrane TYK2 JAK1 TYK2 JAKI JAK1 TAT STAT STAT P P STAT P P STAT1 STAT2 TATI STAT1 STAT STAT P GAR ISGF3 STAT STAT2 D STATI O REC Nucleus Type II ISG VI TRENDS in Immunology JAKZ JAK2 Cytosol JAK1 TAT STAT T1 0

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