Wednesday, July 23, 2014

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 CD45RBhi). 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.


Monday, July 21, 2014

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.


Sunday, July 20, 2014

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.


Sunday, July 6, 2014

Inflammasome immortality

Immune system has many ways to detect the presence of virulence factors. One such system is an assembly of the super-complex called inflammasome. For its proper activity this super-complex requires close association of several components: NALP3, ASC and pro-Caspase 1. Depending on initial trigger, NALP3 can be replaced with NLRP1, AIM2, NLRC4 in inflammasome super-complex. Once formed, inflammasome cleaves pro-IL-1 or pro-IL-18 into mature active proteins IL-1beta and IL-18.

Earlier studies have shown that inflammasome assembly and function occurs in the cytoplasm. Now, two new papers published in Nature Immunology reveal that inflammasome continues to function as a secreted, extracellular template and amplifies local IL-1 production and recruitment of granulocytes.

I am going to discuss the results of several principal experiments from these studies.

In the first paper (1), the authors generated macrophage line expressing ASC molecules fused to fluorescent protein (ASC-FP). In resting state, ASC-FP were evenly distributed in cytoplasm. Upon inflammasome activation, however, ASC-FP formed oligomers. Interestingly, the authors detected substantial amount of ASC-FP extracellularly. This release of ASC was associated with cell death and requires caspase 1 activity. Treatment of LPS-primed ASC-KO macrophage cytosol with recombinant ASC showed that recombinant ASC had pro-caspase-1 and pro-IL-1 processing activity, while LPS+ATP combination did not. These results indicated that exogenously added ASC formed super-complexes with endogenous NALP3 and interacted with pro-caspase 1.

To directly observe this phenomenon, the authors used red fluorescent tagged ASC-mCherry construct. They found that (a) ASC-mCherry was phagocytosed and released inside macrophage's cytosol after lysosomal membrane destabilization and it formed oligomers, (b) as a result, an exogenous ASC-mCherry acted as a danger signal for LPS-primed macrophages inducing IL-1beta secretion, similar to silica or nigericin.

More importantly, using two different fluorescent tagged ASC recombinant proteins, ASC-mCherry (red) and ASC-mCerulean (blue), the authors observed that exogenously added ASC attracted endogenous ASC in super-complexes, acted as a template for further oligomerization.

Finally, the authors conducted several in vivo experiments with recombinant ASC and found that injection of exogenous ASC oligomers induced local inflammatory granulocytes recruitment independent of NALP3, but IL-1 receptor dependent manner (this effect was enhanced in presence of anti-ASC antibody or in presence of serum derived from autoimmune mouse strain). Of note, there is opposite results regarding exogenous ASC ability to induce IL-1beta secretion from ASC-KO macrophages (compare Fig .3f and Fig. 3g versus Fig 5e). Either there is reporting error or in vitro bone marrow derived macrophages behave differently from ex vivo inflammatory macrophages.

In second paper (2), the authors also confirmed that inflammasome components were detected in supernatant within 15 min of macrophage activation and they formed oligomers. They also found that addition of recombinant ASC oligomers to LPS-primed ASC-KO macrophage cell line induced IL-1beta secretion (however this required presence of ATP or NALP3, a slightly different result from the first paper). The difference could be due to use of primary macrophages versus macrophage cell line (or immortalized macrophages) in different series of experiments, am obvious limitation. Indeed, comparison of IL-1beta secretion results in Fig. 3J versus Fig. 4B, clearly indicates that dependency on ATP or NALP3 is a function of difference in cell types or difference in dose of recombinant ASC oligomers.

In summary, these papers showed that during inflammasome induced pyroptosis, dying cells release active complexes of inflammasomes that are able to directly process extracellular pro-IL-1 locally or alternatively these oligomers internalized by local macrophages and act as danger signals in propagating inflammation. Since inflammasome-related mutations are linked to several sterile inflammatory diseases, improved understanding of inflammasome biology could yield more targeted therapeutics.


Friday, July 4, 2014

Aryl hydrocarbon Receptor (AhR) keeps tissue "immune" memory alive

Lipopolysaccharide (LPS) is a part of gram-negative bacteria recognized by TLR4. Excessive activation of immune system by LPS induces sepsis. Earlier studies have shown that exposure to initial small doses of LPS renders the host more resilient (more tolerant) to subsequent exposure to higher doses of LPS. This effect is called LPS tolerance (a process more akin to biochemical adaptation).

The new study (1) published in Nature suggests that aryl hydrocarbon receptor activity controls LPS tolerance. AhR is host molecule that recognize endogenous products of tryptophan catabolism called kynurenines. Tryptophan catabolism in mammals is mediated by enzymes IDO1, IDO2 and TDO2 (this latter enzyme is mainly expressed in liver cells).

The authors examined the effect of primary LPS challenge on wild type, AhR-KO, IDO1-KO, IDO2-KO and TDO2-KO mice. Upon exposure to 10mg/kg of LPS, AhR-KO and TDO2-KO mice showed significantly higher level of mortality compared to wild-type or IDO2-KO mice. Of note, in these experiments, IDO2-KO mice behaved similar to wild-type mice (it appears that IDO2 plays no meaningful function in vivo) This increase in LPS sensitivity of AhR-KO and TDO2-KO mice was still observed when different doses of LPS was used (8-fold and 4-fold increase respectively). Interestingly, unlike at 2h post-exposure, levels of pro-inflammatory cytokines, IL-6, TNF-alpha and IL-1beta were higher in AhR-KO and TDO2-KO mice at 24h post-exposure compared to levels in wild-type mice. An opposite effect was seen with IL-10. AhR-KO and TDO2-KO mice expressed less IL-10 at 24h post LPS exposure.

These results indicated that activity of AhR or production of kynurenines had protective effect on host after primary LPS challenge. Indeed, LPS challenge of wild-type mice treated with TDO2 inhibitor dramatically reduced their survival that was reversed by exogenously administered L-kynurenine. This therapeutic effect of L-kynurenine was abolished in AhR-KO mice.

To examine the role of AhR in LPS tolerance, the authors re-exposed wild-type, IDO1-KO, IDO2-KO mice previously primed with primary dose of LPS. In this setting, only wild-type and IDO2-KO mice survived secondary LPS exposure, while IDO1-KO mice all died by day 10 post secondary LPS exposure. This resilience of wild-type mice to re-exposure to LPS was mediated by AhR activity since its blockade by specific inhibitor cut survival rate of pre-exposed wild-type mice. The authors showed that beneficial effect of AhR was mediated through TGF-beta and reduced expression of IL-6, TNF-alpha, not seen in IDO1-KO mice.

Finally, to see the effect of LPS tolerance in clinically relevant infection model, the authors infected mice with either gram-negative (S. Typhimurium) or gram-positive bacteria (group B Streptococcus). When mice were pre-exposed to LPS prior to infection with bacteria, they showed less immunopathology, reduced pro-inflammatory cytokine production and improved survival in response to infection compared to untreated control mice.

In summary, this study confirmed that the products of tryptophan catabolism activates AhR and contribute to increased tissue tolerance (biochemical adaptation) to secondary exposure to infection or noxious products.