Why is this IL-33 paper so "alarmin"-gly bad?

Some papers just make no sense, especially when they appear in top journals, like Nature. What's going in editor's mind when they are green-lighting this type of research for publication?

Here is a new example for such paper. It was recently published in Nature (1). This study comes from Fiona Powrie's lab in UK. She became well-known for her studies of experimental colitis model in rats or mice upon adoptive transfer of naïve T cells (then, in 90's, called CD45RB^hi). Her research is mainly focused on interplay between naïve T cells and Foxp3+ Tregs in colitis models.

This study is another variation of this approach.

First, the authors made an observation that gut associated Tregs express IL-33 receptor, while few Tregs from spleen or lymph nodes do.

Next, in vitro test showed that combination of IL-33 and TGFbeta-1 augments de novo Foxp3+ Treg differentiation (IL-33 alone had no effect).

To assess in vivo role of IL-33 signaling in Tregs, the authors first generated mixed bone marrow chimera mice using wild-type and IL-33 receptor-deficient marrow cells (also called ST2 -/-). Next, colitis was induced by combination of treatment with Helicobacter hepaticus and anti-IL-10R injection. Analysis of number of colonic Tregs derived from IL-33 receptor-deficient marrow cells (which can not respond to IL-33) were reduced 2-fold compared to wild-type Tregs. In addition, IL-33 receptor-deficient Tregs expressed reduced amount of Foxp3 compared to wild-type Tregs (1000 vs. 800 MFI).

Another set of in vivo experiments showed that IL-33 receptor-deficient Tregs were less potent in preventing colitis induction by naïve T cells. At 8 weeks post transfer, IL-33 receptor-deficient Tregs lost much of Foxp3 expression. Of note, in vitro IL-33 receptor-deficient Tregs were as potent as wild-type Tregs.

To connect this new observation with their previous studies about IL-23 and colitis, the authors showed that IL-23 blocks signaling by IL-33 in Tregs.

Finally, another set of in vivo adoptive transfer experiments into RAG1/IL-23R double knockout mice showed that in even in absence of IL-23 signaling in the host, IL-33 receptor-deficient Tregs were less potent in preventing colitis induction. 

The data in Fig. 4d, however, are difficult to interpret. If IL-23 was blocking Tregs induction solely through IL-33 inhibition, absence of IL-23 should have had two fold outcome:

(a) induced much potent wild-type Tregs and

(b) IL-33 receptor-deficient Tregs should become more potent in preventing colitis

However, neither outcome has been observed, that is, in my opinion, very unusual and unexplainable result and questions the relevance or importance of all other data.

David

Foxp3+ Tregs - IgA - microbiota: the Axis of Good

Foxp3+ T regulatory cells (Tregs) are thought to regulate immune system. But who regulates the regulators? Any biologically functioning regulatory network should be based on feedback mechanism. If tissue regulates Foxp3+ Tregs, then Foxp3+ Tregs should be able to sense the state of the tissue and vice versa.

Alternatively, a gut microbial flora is a good candidate for regulation of Tregs. Based on what we have already uncovered about gut microbiota, I expect that many of our physiological functions will be found to be fine tuned by gut flora (food taste, mating preferences or even aging).

This new paper (1) I am going to review this time was published in Immunity (lots of good papers started to appear in Immunity indicating better editorial control on quality) and came from Sidonia Fagarasan's lab in Japan. She is one of the most interesting and fascinating scientists. Originally from Romania, she made her famed career as immunologist in Japan, quite an extraordinary achievement for a female scientist to do it in that male-dominated scientific circle.

Her lab's main focus is IgA production and its regulation. In recent years, her lab published several papers linking Foxp3+ Tregs with gut IgA production and role of gut flora in all this.

This new paper is a further refinement in that direction. I liked it because it has one very cool and visually effective figure (see big figure below) that simplifies understanding of the complex data about gut microbiome.

IgA is a signature Immunoglobulin of mucosal surfaces. Mucosal surfaces are the critical spaces where our immune system comes in contact with microbes and has to decide what to do.

First, this paper has compared the gut microbiome composition of mice that lack T cells (CD3e -/-) or B cells (Ighm-/-) or both (RAG1 -/-). In all these cases, gut microbiome diversity and phylogenetic structure were affected by the absence of either T or B cells. The authors speculated that it has to do with lack of IgA and/or Foxp3+ T reg production.

To directly examine this hypothesis, the authors used adoptive transfer experiments. Specifically, they transferred naïve T cells or Tregs, separately or together into T cell-deficient CD3e-/- mice. Unlike naïve T cell transfer (that caused colitis), transfer of Tregs alone or with naïve T cells restored microbiome diversity and its phylogenetic structure of recipient CD3e-/- mice to the level seen in wild-type mice (though they don't show wt mice gut flora in this particular graph ). Especially striking effect were seen with bacteria called Firmicutes cluster IV and XIVa and XVIII. These results indicated that not only gut bacteria can influence T reg induction (as previously reported), but Tregs in turn can influence the composition of gut flora.

Interestingly, transfer of Foxp3+ Tregs into T and IgA double deficient mice failed to restore microbiome diversity and its phylogenetic structure, implying that local production of IgA was necessary to mediate Tregs effect on gut flora.

In addition, co-transfer of naïve T cells with Tregs that lacked bcl6 expression (necessary for GC follicular Treg development) also failed to restore gut Firmicutes, while still capable of preventing colitis. This showed that GC function of transferred Tregs was important for gut flora normalization.

Next, the authors tested the effect of Foxp3+ Treg-educated gut flora on naïve germ-free (GF) mice. As expected, transfer of Treg-educated gut flora (basically, feces) from donor CD3-/- mice into recipient ex-GF mice promoted IgM to IgA switch, while naïve T cell-educated gut flora induced IgG1 switch as well. Alternatively, when CD3-/- GF mice initially received Treg- or naïve T cell-educated gut flora and then received Treg cells, T reg-educated gut flora promoted donor Treg expansion, GC and IgA generation, while naïve T cell-educated gut flora lacked this properties. These results showed that T reg-educated gut flora acted as a messenger for further amplification or maintenance of Foxp3+ Treg population.

It would have been interesting to see whether T reg-educated gut flora could suppress colitis induction in CD3-/- ex-GF recipient mice when transferred with donor naïve T cells.

In summary, the data from this paper suggest that Foxp3+ Tregs modify and educate gut flora composition through IgA production, which in turn can amplify gut associated T reg-IgA axis.

David

Why is IgE response so “Syk”-ening?

Allergy is an immunological mystery. Immunoglobulin E, hence IgE, mediates the vast majority of allergic reactions. But what is the evolutionary advantage having such a damaging immune response?

I would like to review new paper (1) from Immunity that examined the role of IgE signaling in a mouse model of human peanut allergy. The data in this article are very straightforward.

The authors used mutant mouse model with hyperactive IL-4 receptor alpha mutation (F709 mutation). This model permits study of human-like food allergic response in mice.

First, the authors found that oral gavage (quite stressful procedure) of mice with peanut butter (4 times, weekly, 5 mg protein, sensitization) induced serum peanut-specific IgE and Th2 response (high IgG1, high IL-4) in IL-4 receptor mutant mice, but not in wild-type mice. In contrast, there was an inverse relationship between peanut-specific Foxp3+ T reg cells proliferation and presence of hyperactive IL-4 receptor alpha mutation. Interestingly, this human-like peanut-specific Th2 response was abolished in peanut-sensitized IgE-deficient mice and correspondingly, peanut-specific Foxp3+ T reg cell proliferation was recovered in peanut-sensitized IgE-deficient mice even on IL-4 receptor alpha mutation background.

Moreover, high dose, 100 mg peanut challenge of peanut-sensitized mice revealed that core body temperature reduction (a readout for anaphylaxis) was observed only with IL-4 receptor alpha mutant mice, but absent in (a) unsensitized IL-4 receptor alpha mutant mice, (b) wild-type sensitized mice or (c) obviously on IgE-deficient background. This results suggested that presence of IgE favored antigen-specific Th2 response and inhibited antigen-specific Foxp3+ T reg cell proliferation.

Since anti-IgE therapy has been developed to treat human allergies, the authors used similar approach with anti-IgE injection and found that anti-IgE injection prior to peanut-sensitization stage or even during desensitization stage (when allergy has already been established) prevented anaphylaxis to subsequent high dose peanut challenge. Desensitization with high dose peanut alone did not prevent anaphylaxis. This results indicated that anti-IgE therapy is a viable immunotherapy.

Next, the authors tested the role of mast cells in peanut allergy model. They have tested 3 different mast cell deficient mouse models: Kit-deficiency (naturally lacks mast cells, among other abnormalities), Mcpt5-cre iDTR, and Mcpt5-cre Syk fl/fl mice models. Mcpt5 is a mast cell specific promoter. Mcpt5-cre iDTR permits specific depletion of mast cells and Mcpt5-cre Syk fl/fl model permits mast cell specific IgE-signaling molecule, Syk inactivation.

The authors showed that Kit-deficient mice on IL-4 receptor alpha mutant background were protected against anaphylaxis. However, reconstitution of Kit-deficient / IL-4 receptor alpha mutant mice with wild-type but not IL-4-deficient mast cells restored anaphylaxis. Similarly, selective depletion of mast cells in Mcpt5-cre iDTR mice or selective inactivation of IgE signaling in mast cells in Mcpt5-cre Syk fl/fl mice prevented anaphylaxis, reduced peanut-specific IgE production and augmented peanut-specific Foxp3+ T reg proliferation. This results showed that mast cells derived IL-4 and mast cell specific IgE signaling plays a critical role in this model of human peanut allergy.

Finally, to test the therapeutic effect of Syk inhibitors, the authors injected allergy-prone IL-4 receptor alpha mutant mice with available Syk inhibitor during either peanut sensitization or desensitization stage. In both cases, peanut application under the cover of Syk inhibitor prevented anaphylaxis. Interestingly, treatment with Syk inhibitor specifically reduced peanut-specific IgE production without affecting IgG1 level. Since application of Syk inhibitor led to peanut-specific Foxp3+ Treg expansion, the authors tested therapeutic effect of the adoptive transfer of Foxp3+ T reg cells from Syk inhibitor treated peanut-sensitized IL-4r mutant mice into untreated peanut-sensitized IL-4r mutant mice. Transfer of Foxp3+ T reg cells from Syk inhibitor treated mice into peanut-sensitized mice reduced IgE and prevented anaphylaxis.

In summary, this paper showed that mast cell-specific IL-4 production and mast cell-specific IgE signaling through Syk plays critical role in mouse model of peanut allergy. This allergic reaction however could be prevented by anti-IgE and Syk inhibitor therapy or by antigen-specific Foxp3+ Treg transfer.

There are few good new papers about Foxp3+ T reg cells recently published in high impact journals and I will review some of them next.

David