Thursday, December 31, 2015

IL-2 instructs allergen-specific tissue resident memory TH2 cell development

IL-2 is one of the first cytokines [interleukins] discovered and we still have no clear idea of the extent of its involvement and role within immune system. One reason has to do with the fact that IL-2 "in vitro" and IL-2 "in vivo" behave if completely 2 different cytokines.

So, if I see new paper that could tell us more about IL-2, I can't resist reading it. This week journal Immunity has published one such article. There, the authors showed that IL-2 signaling via its high-affinity receptor IL-2α (also known as CD25) directed development of allergen-specific tissue resident memory TH2 cells. I will discuss only those data that are relevant and unequivocal for the story.

For this study, the authors used class II tetramers and i.v. labeling techniques to identify and track house dust mite (HDM) allergen-specific tissue resident T cells. They showed that primary allergen exposure generates lung tissue resident HDM-specific tetramer-positive CD4 T cells.

These lung tissue resident tetramer-positive CD4 T cells expressed IL-13 upon HDM re-challenge (that identified them as TH2 cells).

Next, using parabiont mouse model the authors showed that tetramer-positive CD4 T cells found in allergen challenged lung tissue were bona fide non-circulatory resident-memory TH2 cells.

Finally, using WT:CD25KO mixed bone marrow chimera mice the authors showed that development of lung tissue resident memory TH2 cells required IL-2 signaling via its high-affinity receptor IL-2α.

In summary, this study suggests that IL-2 signaling via its high-affinity receptor IL-2α is mandatory for resident-memory TH2 cell development. This is addition to already known IL-2 roles in Foxp3+ Treg and memory CD8 T cell development. From therapeutic point of view, this is one big mess.

David Usharauli

Wednesday, December 30, 2015

Steady-state IL-12p70-producing CD103+ DCs control magnitude of type II immunity

In my previous post I reviewed the role of microbiota and MyD88/TRIF pathway in CD103DC-derived TGF-β dependent IgA production. In the following review I will continue analyzing immunobiology of CD103DCs. Surprisingly, new paper published in JEM claims that CD103+ DCs naturally producing biologically active IL-12p70 restrain type II immunity during worm infection.

I have done some work on IL-12, so I was curious to review this paper. So lets begin. I will only show the data that I believe are relevant for the story. In first set of experiments, the authors showed that Batf3 -/- mice that lack CD103DCs have exaggerated type II response to S. mansoni eggs and worm infection itself (a typical Th2 trigger).
Similar exaggerated Th2 response were observed in Batf3 -/- mice infected with another Th2 trigger H. polygyrus. 

More importantly, Batf3 -/- mice showed increased resistance to H. polygyrus infection. 

To understand how Batf3-dependent CD103DCs influenced Th2 response, the authors analysed expression of IL-12, a cytokine known for its role in inhibiting Th2 response. Indeed, Batf3-dependent CD103DCs were main producers of steady-state IL-12 as measured by YFP expression under p40 promoter (p40 is a β chain of heterodimeric IL-12p70).

Finally, using bone marrow chimera, the authors showed that lack of biologically active IL-12 (IL-12p70) derived from Batf3-dependent CD103DCs was indeed responsible for exaggerated [and protective] Th2 response of Batf3-/- mice to H. polygyrus.

Of note, in separate set of experiments with germ-free and MyD88/TRIF DKO mice, the authors claim that Batf3-dependent CD103DCs were producing biologically active IL-12p70 independently of microbiota or TLR signaling. However, in contrast to YFP expression, flow staining pattern with antibody against p40 revealed little staining of IL-12p40 in CD103DCs from any of the mice analyzed. So, if not the data with IL-12p35-/-/Batf3-/- BM chimera, I would have dismissed this whole study as an artifact (to be convincing the authors need to show these results with p40-YFP mice on GF and MyD88/TRIF background).

In summary, this study suggests that Batf3-dependent CD103DC-derived IL-12p70 influences magnitude of type II immunity.

David Usharauli

Tuesday, December 29, 2015

Microbiota drives airway IgA class switch via DC-derived TGF-β

IgA is a class of antibody specifically designed for protection and tolerance at mucosal surfaces. Since vast majority of antigens [both infectious or noninfectious nature] interact with mucosal tissues, knowledge of signals controlling IgA production would help to develop more robust vaccination protocols.

So I decided to review this paper from Journal of Experimental Medicine where the authors have analysed IgA promoting capacity of lung DCs.

Most experiments were done ex vivo on sorted DC and B cells. Most cultures included α-IgM and α-CD40 antibody to "mimic" T-dependent Ab production. First, the authors noticed that lung CD103+ and CD24+ DCs (but not CD64+ Mφ) could provide necessary signals to naive B cells to switch to IgA (similar to MLN DCs).

Next, the authors found that this IgA switch capacity of lung CD103+ and CD24+ DCs  were dependent of their ability to produce TGF-β and Retinoic Acid (Vitamin A metabolites).

Next, the authors found that microbiota influenced lung DCs capacity to drive IgA switch in naive B cells [though it is not clear whether it is airway or gut microbiota that does it].

Follow up experiments revealed that MyD88/TRIF signaling [most likely from microbiota] in lung DC cells increased TGF-β production and their IgA switch potential.

Finally, the authors showed that i.n (intra-nasal) or i.t. (intra-tracheal) immunization [but not s.c. immunization] with small dose of cholera toxin (CT) provided system-wide protection against cholera toxin re-challenge, implying body-wide re-distribution of airway primed CT-specific B cells.

In summary, this study showed the role of microbiota/MyD88/TGF-β/CD103+ DCs axis in driving airway mucosal IgA class switch.

David Usharauli

Wednesday, December 23, 2015

DNA vaccine immunogenicity requires STING but not cGAS activity

Intracellular DNA sensor STING has become a major player in mediating the range of immune responses from viruses to tumors to autoimmunity.

From practical point of view, a therapeutic application of STING pathway would involve development of DNA vaccines both for infectious diseases or cancers. In general, DNA vaccine has a long history of testing, but if I am not mistaken there is still no FDA-approved human DNA vaccine [though I think there is approved DNA vaccines for domestic animals].  

So it was interested to read this new paper in Journal of Immunology where the authors found that priming of adaptive immune response by DNA vaccine expressing Flu virus H1 antigen required STING but surprisingly not cGAS pathway.

This is a simple paper. First, the authors found that two i.m injections of H1HA DNA vaccine could prime anti-Flu H1-specific CD8 T cell and IgG response in WT but not in STING KO mice.

However, surprisingly, cGAS KO mice that are deficient of enzyme upstream of STING pathway showed normal response to this DNA vaccine.

Finally, mice deficient for IRF7, but not IRF3, showed similar [to STING KO] reduction of DNA vaccine-induced adaptive immune response (earlier this year journal Science has published research article describing IRF7 deficient patient who showed severe susceptibility to H1N1 viral infection).

In summary, this paper showed that DNA vaccine immunogenicity required STING/IRF7 pathway rather than classical cGAS/STING/IRF3 pathway.

Of note, since the authors have not done virus challenge experiment, the results in this study do not necessarily tell whether this DNA vaccine was effective in providing clinically relevant protection against Flu virus [one of the reasons this study was published in Journal of Immunology and not in a more prestigious journal, in my opinion].

David Usharauli

Monday, December 21, 2015

Spontaneous death of myelin-producing oligodendrocytes could trigger delayed MS-like symptoms

I usually don't check Nature Neuroscience. It does not typically publish immunology-related studies [though MS studies are of interest]. So I was surprised to see such "heavily" packed immunology article there and decided to review it to understand how it ended up there.

It is immediately clear that is not written from immunologist point of view. In general, inter-disciplinary studies are encouraged but if it is not done properly it produces lesser quality research

To this end, the authors have used Plp1-Cre-ERT;ROSA26-eGFP-DTA mouse model where tamoxifen injection releases stop signal from diphtheria toxin A production in oligodendrocytes leading to their death. It appears that this is a rare model of oligodendrocyte deletion where mice actually survive long-term (but it appears this DTA model show late-onset [starting at weeks 26] "leakiness" in absence of tamoxifen injection. This knowledge in itself creates host of issues in data interpretation).  

So, the authors noticed that starting 40 weeks post tamoxifen injection [but not at 10 weeks], spleen and cervical lymph nodes of Plp1-Cre-ERT;ROSA26-eGFP-DTA mice contained MOG-specific effector T cells. These were accompanied with clinical symptoms of EAE.

Similar results were found with 2D2 transgenic CD4 T cells [specific for MOG] transferred into tamoxifen-treated Plp1-Cre-ERT;ROSA26-eGFP-DTA host.  

Now, next experiments were quite surprising. To clearly show the role of T cells in the development of late onset MS-like symptoms in tamoxifen-treated Plp1-Cre-ERT;ROSA26-eGFP-DTA mice the authors tried to cross this DTA model with RAG KO mice. Interestingly, these T/B cell-deficient DTA mice did not survive after tamoxifen injection, implying that recovery from acute oligodendrocyte deletion [following tamoxifen injection] somehow required presence of T or B cells [reminds of studies done by Michal Schwartz lab]. However, the authors neither tried to use CD3KO or B cell KO or simply Ab depletion to test these hypotheses.

Other set of experiments with adoptive transfer of T cells harvested from tamoxifen-treated Plp1-Cre-ERT;ROSA26-eGFP-DTA mice into RAG deficient mice (but not in WT host) produced MS-like symptoms.

In the remaining experiments the authors tried to show that injection of MOG peptide coupled to nanoparticles could tolerize self-reactive T cells.

So what we learned from this study? First, I am surprised that it even get into Nature Neuroscience [it does not belong there]. Now, this could mean few things: (1) this research was rejected from Nature Immunology and ended up in Nature Neuroscience; (2) It was directly sent to Nature Neuroscience but reviewed by non-immunologists; (3) standards for Nature Neuroscience is much lower compared to Nature Immunology, in general.

David Usharauli

Friday, December 18, 2015

Both TNF-α and IL-1 are necessary for M. tuberculosis control

Both TNF-α and IL-1α are innate cytokines. For both cytokines there are FDA-approved biological drugs such as Humira® (TNF-α blocking) and Kineret® (IL-1RI blocking). Of note, anti-TNF-α, but not anti-IL-1RI blockers, carry specific FDA label regarding M. tuberculosis incidence as a drug side effect. 

So I was surprised to read new paper in journal Immunity where the authors showed that in mouse model of aerosol M. tuberculosis infection both TNF-α and IL-1 were required for efficient control of TB pathogen.

Of note, this article was under review for more than two years. I guess it took long time to convince editors to publish it. The study is based on analyses of bilateral bone marrow reconstituted knockout mice. Basically a descriptive study. I modified sequence of figures for clarity.  

In a first set of experiments the authors showed that mice deficient for IL-1α and IL-1β signaling on hematopoietic cells (DKO or IL-1RI KO) were highly susceptible to M. tuberculosis infection due to exaggerated, non-productive inflammatory response.

Follow up experiments with mice single deficient for IL-1α and IL-1β confirmed physiological significance of individual IL-1 molecules in host's defense against M. tuberculosis infection (the authors claim that IL-1β deficient hosts are less susceptible to M. tuberculosis infection, but overall survival trend is the same).

Significance of IL-1α in control of M. tuberculosis infection was supported by observation that transfer of stem cells expressing viral-encoded IL-1α under the control of CD11c promoter restored IL-1α KO host's resistance to M. tuberculosis infection (why the authors have not done the same experiment with IL-1β is unknown).
Next, the authors showed [or rather re-confirmed] that mice deficient for TNF-α signaling on hematopoietic as well as on non-hematopoinetic cells were susceptible for M. tuberculosis infection.

Additional experiments revealed that absence of signaling via both IL-1RI and TNF-R1 on non-hematopoietic cells [but not on hematopoietic cells] further diminished host's resistance against M. tuberculosis infection.

In summary, this study indicates that in mouse model of M. tuberculosis infection both TNF-α and IL-1 molecules play non-redundant role in host's defense. Interestingly, mice individually deficient for TNF-α or IL-1 signalling on hematopoinetic cells showed similar susceptible to M. tuberculosis infection (in contrast, only mice deficient for TNF-α, [but not IL-1] signalling on non-hematopoietic cells retain susceptibility to M. tuberculosis infection).

However, how these data fit with clinical studies with Humira® and Kineret® is another story altogether. Surprisingly, the authors failed to discuss it at all. That's a big problem.

David Usharauli

Thursday, December 17, 2015

Enigmatic tuft cells residing in intestine constitutively produce type II immunity primer cytokine IL-25

Type II immunity is responsible for such physiological and pathological conditions as allergy, anti-parasite [anti-worm] expulsion response, thermoregulation and lean body metabolism. Right now our therapeutic toolkit to influence this system is minimal or even nonexistent. We just don't know enough about it [unlike TH1 response].

This new paper in journal Nature is a good example for this scientific gap in type II immunity. Here, the authors, led by Richard Locksley at UCSF, revealed that little known tuft cells residing in intestine contribute to type II immunity by secreting IL-25.

It appears that within intestine we have 5 cell types, 1 absorptive enterocytes, and 4 secretory cell types: paneth, tuft, goblet and enteroendocrine. By using Flare25 mouse knock-in/deleter model, the authors found that in normal mouse intestine, IL-25 was expressed by rare cells that were also positive for EpCam+ and doublecortin like kinase 1 (DCLK1+). These were markers for tuft cells (EpCamDCLK1+) [IL-25+cells were negative (a) for chromogranin A, a marker for enteroendocrine cells, (b) lysozyme, a marker for paneth cells, and (c) mucin 2, a marker for goblet cells].
Next, the authors showed that tuft cells undergo expansion following worm challenge (type II immune response) in a IL-13-dependent manner (produced by group 2 innate lymphoid cells ILC2).

IL-13 producing intestinal ILC2 were in turn supported by IL-25+ producing tuft cells in a forward-feed circuit.

Physiological significance of IL-25+ tuft cells became evident when the authors showed delay in anti-worm expulsion response in mouse model of epithelial [tuft]-selective deficiency of IL-25 production.

In summary, this study uncovered immunological role of tuft cells in type II immunity. Naturally occurring tuft cell-derived IL-25 support development and maintenance of naturally occurring IL-13+ ILC2 and vise versa.

David Usharauli

Wednesday, December 16, 2015

Comparison of T cell subset tissue-wide distribution during human ontogeny

The authors have established an protocol allowing access to pediatric and adult human donor tissues for research purpose. For each tissue obtained, a detailed overview of T cell subset distributions were generated. 

These experiments yielded following data: 

(a) Early-life human tissues were dominated by naive CD45RA+CCR7T cell subset. Correspondingly, few effector memory CD45RA- CCR7- TEM were detected at this developmental stage (2 months - 2 years).

(b) Functionally, early-life human T cells were less responsive to TCR stimulation (both to anti-CD3 or PMA) compared to adult T cells.

(c) Early-life human tissues contained high frequency of Foxp3+ Tregs.

(d) The high frequency of Foxp3+ Tregs in early-life human tissues was physiologically relevant since their depletion in an in vitro culture restored T cell proliferation (Note, TREG:TEM ratios in tissues from pediatric donors were lowest in lung and small intestine mucosal tissues, places with natural high-level antigen exposure).

In summary, correct understanding of T cell subset distribution throughout the human body may help to implement more productive vaccination protocols.

David Usharauli

Tuesday, December 15, 2015

Collapse of cholesterol biosythesis triggers universal anti-viral response

New study in journal Cell may provide mechanistic clue how obesity [and hyper-cholesterolemia] affect immunity. The authors showed that limiting cholesterol biosynthesis automatically triggers universal anti-viral defense state, mimicking natural response to viruses

Initially, the authors studied effect of type I IFNs [and its triggers] on lipid metabolism in macrophages. These pilot experiments reveal that de novo synthesis of cholesterol was inhibited by type I IFNs and its triggers, such as virus or PolyI:C. Interestingly, total cholesterol level or its import were not affected.

To decouple direct involvement of host defense response in lipid metabolism, the authors used LysM-Cre-Scap fl/fl mice that show limited lipid biosynthesis in myeloid cells. Indeed, Scap-/- macrophages displayed the same decrease in cholesterol synthesis as type I IFN treated control cells.  

Reduction of cholesterol synthesis in Scap-/- macrophages was accompanied with increased resistant to viral infection [similar resistance were observed in MKV (mevalonate kinase) and  HMG-CoA reductase deficient cells that have limitation in cholesterol biosynthesis].

This resistance in Scap-/- macrophages was transferable to control cells via soluble component in conditioned medium (type I IFNs).

Notably, the authors showed that anti-viral resistance in Scap-/- macrophages could be inhibited by exogenous free cholesterol.

Finally, the authors showed that DNA sensing STING-cGAS pathway was responsible for increased anti-viral state that accompanied collapse of cholesterol synthesis, and here too, free cholesterol supplementation was able to inhibit cGAS activity by its ligand [mechanism is unknown].

In summary, this study raises some of the important questions related to host metabolic shift observed during infections. Inhibition of anti-viral state by free cholesterol could explain diminished immune state observed in obese individuals. In addition, there are studies showing how cholesterol pathways, such as geranylgeranylation could influence TH17 and Foxp3+ Treg balance.

David Usharauli

Sunday, December 13, 2015

IL-22 promotes intestinal integrity via direct effect on stem cells

Initially, the authors showed that IL-22 produced by group 3 innate lymphoid cells (ILC3) promoted in vitro growth of intestinal organoids (small intestine crypt cells cultured with EGF, Noggin and R-spondin-1, ENR).

Growth augmentation by IL-22 was mediated via STAT3 signaling, as shown by growth defect of STAT3 KO crypt cells cultured with IL-22.

Unlike Paneth cell depletion, experiments with  Lrg5+ stem cell depletion confirmed that IL-22 promoted intestinal regeneration via its effect on stem cells (though I don't know whether organoids growth could happen without  Lrg5+ cells, in the first place).

Finally, the authors conducted in vivo experiments with rh IL-22 to validate hypothesis that IL-22 or its analog could improve survival in GvHD (condition that leads to intestinal damage). Indeed, treatment of mice with stabilized rhIL-22 analog, F-652, following bone marrow transplantation (BMT), reduced intestinal damage and improved host survival (though mrIL-22 effect was less dramatic in Fig 3).

In summary, this study suggests that IL-22 could provide therapeutic benefits in medical conditions where gut integrity is compromised.

Note: the authors claim that they filed a provisional patent application "on the use of ISC growth factor". I don't believe one could get any patent on naturally occurring products, such as IL-22. In addition, idea that IL-22 affects gut epithelia is well established concept. While the authors refined this concept and showed that IL-22 may work directly on stem cells, this by itself is not a patent-eligible innovation, in my view.

David Usharauli

Friday, December 11, 2015

Autophagy-independent role for Atg5 in M. tuberculosis infection

This is a second paper about neutrophils in Nature this week (advanced online publication). Quite interesting and surprising. It showed that autophagy molecule Atg5 has unique [autophagy-independent] role in protecting against neutrophil-driven immunopathology during M. tuberculosis lung infection.

Initially, the authors showed that myeloid cell-specific deletion of Atg5 (LysM-Cre Atg5fl/fl) made mice highly susceptible to M. tuberculosis infection (that was expected based on earlier studies).

Unexpectedly and surprisingly, however, mice singly deficient for other autophagy components showed normal response to M. tuberculosis infection, implying unique, autophagy-independent role for Atg5 during M. tuberculosis infection.    
Indeed, LysM-Cre Atg5fl/fl mice showed more severe M. tuberculosis-associated immunopathology and neutrophil infiltration (even though LysM-Cre Atg5fl/fl mice did not harbor substantially more M. tuberculosis).
Furthermore, LysM-Cre Atg5fl/fl mice depleted of neutrophils were protected from lung pathology during M. tuberculosis infection.

Finally, neutrophil-specific deletion of Atg5 (MRP8-Cre Atg5fl/fl mice) confirmed pathological role of Atg5-deficient neutrophils during M. tuberculosis infection. 

In summary, this study provides evidence for autophagy-independent role of Atg5 deficiency in neutrophils during M. tuberculosis infection. It appears that neutrophils recruited to the sites of M. tuberculosis infection contribute to tissue pathology rather than provide protection in absence of Atg5. Earlier it was assumed that Atg5 played a protective role during infection purely via its involvement in autophagy formation. However, this new study point to a more complex role of Atg5.

David Usharauli

Thursday, December 10, 2015

Neutrophils prepare niche in the lung for breast cancer metastasis and colonization

Tumors are known to hijack and exploit body's normal function for its own purpose. This is especially true for immune system which often is a reverse-target for growing tumors

Two reasons why this study was accepted in Nature: (1) to promote newly established The Francis Crick Institute based in London [Nature is a UK journal], (2) positive results with Zileuton (Zyflo®), an orally active inhibitor of 5-lipoxygenase and leukotrienes (LTB4, LTC4, LTD4, and LTE4).

First, using mouse model, the authors [it has just 2 authors, very rare] showed that metastasis of breast cancer cells to the lung were supported by neutrophils (genetic depletion of neutrophils in G-CSF-/- or Ela2-Cre-DTA mice as well as antibody-mediated neutrophil depletion reduced tumor metastasis).

Next, the authors found that neutrophil-conditioned medium supported tumor growth in vitro.

It turned out that this tumor-promoting neutrophil-conditioned medium contained high levels of leukotrienes, an active lipid metabolites of 5-lipoxygenase enzyme pathway
Finally, using 5-lipoxygease deficient mouse or Zileuton (Zyflo®) treatment, the authors showed that blocking leukotrienes activity reduced lung metastasis (though Zileuton (Zyflo®) treatment was surprisingly more effective compared to KO mouse).

In summary, this study suggests that anti-leukotriene therapy already available in the market could provide anti-cancer benefits.

David Usharauli