Mystery Rays from Outer Space

Last week I talked some general issues about autoimmunity, and gave a brief background on NKT cells. Today I’ll talk about the paper that spawned that discussion.¹

A common general model for autoimmune goes something like this:

• If you have a genetic predisposition toward autoimmunity²
• And you are exposed to a microbial antigen,



• That is somewhat similar to one of your body’s own antigens
• And the exposure involves inflammation, which sends a “Danger!” signal to the immune system,
• Then immune cells that are normally tolerant to the self antigen
• Become reactive toward the microbial antigen
• And cross-react with the self antigen. This low-level self-reactive inflammation
• Causes cell death, releasing more antigen in the the presence of cell-death “Danger” signals.
• Causing a runaway feedback loop that results in outright autoimmune disease

But as I said, it’s been very difficult to track through a reaction from beginning to end, to support or refute this model.

Matter et al., Fig 3 (Inflamed bile duct)

Primary biliary cirrhosis (PBC) is an autoimmune disease³ of the liver characterized by inflammation of the bile ducts (here is the American Liver Foundation’s PBC information page). The immunity seems to be mainly targeted at mitochondrial antigens, which raises the question of why the liver is specifically involved — mitochondria are found in just about every cell type.

NKT cells recognize CD1, which binds to lipid-type antigens typical of bacterial cell walls. Bendalac’s group found that they could cause a PBC-like disease in mice by infecting them with a particular bacterium⁴ that is normally considered to be a fairly innocuous commensal. They tested this bacterium because it was previously shown to trigger antibodies that cross-react with the mitochondrial antigens that are targets in PBC. (Remember that mitochondria are historically extremely symbiotic bacteria, so the cross-reactivity doesn’t come completely out of the blue.)

Antibodies are produced by B cells. However, the disease could be blocked by preventing NKT cells from getting activated (by infecting mice lacking the NKT target, CD1). The rationale for doing this experiment was that innate immune responses to this particular bacterium are, a little unusually, normally driven by NKT cells.

The autoimmune-type disease lasted in these mice long after they had eliminated the bacteria — months, compared to a week or two to eliminate the actual infection. What’s more, even though NKT cells were essential to get the disease going, once it had started up, the disease could be transferred to new mice by swapping across classical T cells only (i.e. T cells but no NKT cells) — even into mice that had never seen the bacteria and didn’t even have CD1, which were doubly protected against having the disease start on its own. In other words, NKT cells start the disease, but don’t keep it going.

So what seems to be happening is that the NKT cells recognize the bacteria and produce massive inflammation. Because NKT cells tend to home to the liver⁵, they are able to overcome tolerance of cross-reactive cells in the liver, making liver antigens more at risk. The cross-reactive T and B cells, enraged by the constant roar of inflammation the NKT cells produce, attack the cross-reactive self antigens, damaging the cells and causing a constant inflammatory trigger. At this point the disease has become self-perpetuating, and you don’t need the NKT cells any more (and indeed, they quiet down about this time, as the bacteria are eliminated).

These findings establish the missing connection between the microbial innate immune trigger and chronic effector T and B lymphocyte attack of small bile ducts observed in PBC. ⁶

This is probably not a universal effect in detail — NKT cells are likely not important in the majority of autoimmune diseases — but it does give support to the general concepts that have been floating around for a while now.

1. Mattner, J., Savage, P., Leung, P., Oertelt, S., Wang, V., Trivedi, O., Scanlon, S., Pendem, K., Teyton, L., Hart, J. (2008). Liver Autoimmunity Triggered by Microbial Activation of Natural Killer T Cells. Cell Host & Microbe, 3(5), 304-315. DOI: 10.1016/j.chom.2008.03.009[↩]
2. Usually the mechanism is unknown[↩]
3. Probably. There us still some uncertainty, but that is the best bet[↩]
4. Novosphingobium aromaticivorans[↩]
5. For reasons that are not, as far as I know, understood[↩]
6. Invariant Natural Killer T Cells Trigger Adaptive Lymphocytes to Churn Up Bile. Sebastian Joyce and Luc Van Kaer. Cell Host & Microbe (15 May 2008) 3:275-277[↩]

We walk a fine line between death due to immune deficiency, smothered under the weight of pathogens and parasites, and death by hyperimmunity, eaten alive by our own defenses. It’s amazing that our immune system can be tuned so precisely as to recognize anything foreign, yet ignore the vast antigenic universe of our own normal self.

Of course, sometimes the immune system fails, in both directions. We often hear about deaths from pathogens, and autoimmune diseases in general are pretty common. There are many ways by which (it’s believed) the immune system can become self-reactive, but a very common observation is that there are both genetic and environmental predisposing causes to autoimmunity. That is, you may have the genetic makeup to be autoimmune, but until you’re exposed to some environmental trigger, autoimmunity never develops. So, for example, if your identical twin has an autoimmune disease, you are much more likely than someone in the general population to develop the disease; but you still have a good to excellent chance of never getting the disease.

In many cases the neither the environmental triggers nor the genetic factors are well understood. The most likely environmental trigger, though, is some kind of microbe. In some cases, this may be because of “molecular mimicry” — the microbe has an antigen that looks like self antigen; the self antigen is normally ignored, because the immune system needs some kind of “danger” signal before it becomes activated; the microbial antigen is seen in the context of microbial “danger” signals; an immune response forms against the microbial antigen; the immune response cross-reacts with the self antigen; self cells are damaged by this immune response; the dead cells release more danger signals along with self antigen; and a positive feedback loop drives a full-fledged autoimmune disease.

That’s the model, but there aren’t many, if any, diseases where the whole process has been tracked through step by step; in fact, I think that there has been so much difficulty getting clear molecular connections between microbes and autoimmunity that there’s a robust search for other mechanisms. However, in the latest issue of Cell Host and Microbe, Albert Bendelac’s group shows a series of links between bacterial infection and the autoimmune disease human primary biliary cirrhosis (PBC).¹ (There’s also a helpful, if rather dry, commentary² by Sebastian Joyce and Luc van Kaer in the same issue.) Rather than trying to cover everything today I’m going to give background here, and then talk about the specific findings in a few days.

One interesting thing about Bendelac’s paper is that they link CD1 to the disease, through NKT cells. CD1 is an MHC class I family member; I talked about it back here, and that’s its mug shot to the left here (click for a larger version). CD1, like many members of the MHC class I family, has a “groove” in its “top” side. MHC class I proper binds peptides in that groove, but CD1 has a much more hydrophobic groove that binds to greasy things like lipids, glycolipids, and lipopeptides. These kinds of molecules are typically found in some kinds of bacteria — especially mycobacteria, like tuberculosis and leprosy, but also other kinds of bacteria such as the commensal microbe Sphingomonas.

MHC class I molecules, with their peptides, are recognized by cytotoxic T lymphocytes (CTL),³ but CD1 molecules and their lipids are recognized by a specialized subset of T cells, “natural killer-like” T cells (NKT cells). The function of this CD1/NKT system really isn’t all that clear. The early guesses that this was a branch of the immune system specialized for dealing with mycobacteria has been weakened as NKT cells have been linked to resistance to various viruses, and also as various viruses have been shown to block CD1 — suggesting that CD1 and NKT cells would otherwise eliminate them.

OK, enough for now. In my next post I’ll talk more about the disease itself, and then try to spell out the process by which, according to Bendelac, NKT are central to the autoimmune reaction; as well as how this abnormal reaction suggests some of the normal functions of NKT and CD1.

1. Mattner J, Savage PB, Leung P, Oertelt SS, Wang V, Trivedi O, Scanlon ST, Pendem K, Teyton L, Hart J et al. (2008) Liver Autoimmunity Triggered by Microbial Activation of Natural Killer T Cells. Cell Host & Microbe 3:304-315.[↩]
2. Joyce S, Van K, Luc (2008) Invariant Natural Killer T Cells Trigger Adaptive Lymphocytes to Churn Up Bile. Cell Host & Microbe 3:275-277.[↩]
3. And natural killer cells, but let’s not go into that now[↩]

“Human cytomegalovirus infected human endothelial cells” by Joerg Schroeer

There’s a famous picture in Field’s Virology¹ showing how ectromelia (mousepox virus, a model for smallpox) infects a new host, spreads within the mouse, and then is transmitted to a new host. The figure is below² (click for a larger version). Simplified, ectromelia initially infects the skin through small cuts; it replicates at the site, then spreads through blood and lymph to organs (spleen, liver) where it replicates further. The progeny virus from this replication then spreads again through the blood, this time back to the skin, where it replicates once again (now vastly amplified from the initial infection) to form the classic “pock” lesions, which shed virus that can infect a new victim.

It’s generally accepted that this is a common pattern of pathogenesis for many of the viruses that go systemic; not necessarily all viruses, because certainly some remain localized or only spread through, say, direct contact, but for something that spreads through the entire host, it should be a reasonably accurate model.

Human and mouse cytomegaloviruses certainly spread throughout the entire host, and infect many cell types within the body — endothelial cells, lymphoid cells in the spleen and elsewhere, liver, and probably other tissues as well. However, Ulrich Koszinowski’s group now suggests that in spite of this, it isn’t following the ectromelia pattern; replication within some of the organs (liver) is a dead end, that doesn’t help disseminate the virus. ³

Koszinowski has a habit of constructing very cool systems for analyzing his pet virus; sometimes so fancy that I wonder if he makes them a little baroque just because he can (and I know⁴ that he has occasionally been bitten by his elaborate systems). Here he used a Cre/lox recombination system, with the flox in the CMV and the Cre in the mouse under cell-specific promoters, so that the virus genome gets modified only when it replicates in the particular organ. Don’t worry about the details, the point is that the virus is tagged as soon as it replicates in a particular organ, so you can look at viruses throughout the whole mouse and identify whether their ancestors ever replicated in one particular organ. You can also work out timing of replication, and a few other things.

The unexpected bottom line is that what happens in the liver, stays in the liver. There is more MCMV in the liver than anywhere else in the body, but it’s a dead end; once it’s in the liver it doesn’t spread to other organs.⁵ Instead, the relatively small amount of virus that replicates in endothelial cells (and perhaps in the spleen) seems to be a major source for further spread within the body and for transmission.

The results challenge the concept that organs that produce the bulk of infectious virus during acute infection necessarily also play a major role in dissemination.

Surprisingly, to me anyway, even suppression of the adaptive immune system didn’t change this; the virus still hung out in the liver. (The door is still open for innate immune restriction of spread.)

This shows how little we really know about what goes on in authentic viral infections. So much of our understanding of virology is based on tissue culture, but it’s harder than it looks to extrapolate a simple in vitro observation to the complicated interactions within the body, and it’s dangerous to extrapolate from one virus to another.

Why does CMV replicate in the liver if it’s a dead end? Is this simply a matter of indifference to the virus (replicate anywhere you can and hope you’re in the right spot to spread) or is it doing something specific to modulate the host in some way? My bias is that this is something the virus is doing for a reason, but I don’t know what.

1. It’s adapted from a figure in Fenner’s Viral Pathogenesis, which I haven’t read:
   Fenner F, Buller RM. Mousepox. In: Nathanson N, Ahmed R, Gonzalez-Scarano F, et al., eds. Viral Pathogenesis. Philadelphia: Lippincott-Raven; 1997:535-553.
   and is based on old research from Fenner:
   Fenner, F. (1949). Mouse-pox; infectious ectromelia of mice; a review. J. Immunol. 63, 341-373.[↩]
2. I think this qualifies as fair use[↩]
3. Sacher, T., Podlech, J., Mohr, C. A., Jordan, S., Ruzsics, Z., Reddehase, M. J., and Koszinowski, U. H. (2008). The major virus-producing cell type during murine cytomegalovirus infection, the hepatocyte, is not the source of virus dissemination in the host. Cell Host Microbe 3, 263-272.[↩]
4. That is, “I have heard rumors that … “[↩]
5. Even though the virus in the liver is actually perfectly competent for replication in other tissues, if taken from the liver and used to infect other mice.[↩]