IL-17 response to airborne fungi are driven by gut commensal pathobiont C. albicans

A new paper in @CellCellPress from Bacher/Scheffold lab showed that IL-17+ T cell response to airborne #fungus such as #Aspergillus fumigatus found in individuals with #asthma,#COPD, and CF,are in fact expanded by gut commensal pathobiont #Candida #albicans via #crossreactivity.

— David Usharauli (@3DiMMUNE) February 23, 2019

1. majority of IL-17+ #Tcells found in healthy donors are specific C. albicans. #Immunology #TH17 #fungi pic.twitter.com/3WITidrcCn

— David Usharauli (@3DiMMUNE) February 23, 2019

3. In fact, when C. albicans-specific T cells were depleted from #Aspergillus fumigatus-specific T cell pool IL-17 response was gone too suggesting that most if not all of IL-17+ T cells specific for A. fumigatus were indeed #crossreactive to C. albicans. pic.twitter.com/cZEu50Uwep

— David Usharauli (@3DiMMUNE) February 23, 2019

5. similar expansion of Aspergillus fumigatus-specific T cells producing IL-17 were observed in individuals with #crohnsdisease too. pic.twitter.com/L0VbmCcFj5

— David Usharauli (@3DiMMUNE) February 23, 2019

As both #fungi are naturally available in the environment any #immunopathology driven by excess of IL-17 response must be the result of some other changes in the host that makes it susceptible to pathology. what???

— David Usharauli (@3DiMMUNE) February 23, 2019

CD4 T cells silence innate over-activation to gut microbiota

A simple but very effective study was published in journal Nature from Ron Germain's lab. His group is known for publishing high quality in situ microscopy data combined with cellular analysis. New study continues this trend. 

In this study the authors analyzed pattern of phosphorylation of STAT3 transcription factor in small intestine derived from various immune deficient mouse strains. Compared to WT mice, pSTAT3 staining in RAG1 KO gut tissue (lacking adaptive immune system) was significantly up-regulated. 

Analysis of antibiotic-treated or germ-free mice indicated that pattern of pSTAT3 staining in RAG1-KO was correlated with the presence of gut microbiota.

Interestingly, longitudinal analysis showed that pSTAT3 staining inversely correlated with  maturation of adaptive immune system post weaning (between 4-20 weeks). 

Co-housing experiments showed that T cells, but not B cells, played a role in silencing innate pSTAT3 over-activation.

And out of T cells, it were CD4 T cells and class II antigen-presentation that played the role in pSTAT3 silencing.

Finally, both Tregs and SBF-specific Th17 cells (7B8 transgenic T cells) could mediate silencing of pSTAT3 over-activation. Both T cell type could down-regulate STAT3 phosphorylation in innate and epithelial cells but the mechanisms could be different. 

In summary, this study showed that persistent pSTAT3 over-activation observed in mice deficient for CD4 T cell function could explain some of chronic metabolic shifts observed in clinical settings. 

posted by David Usharauli

Salt-sensitive lactobacilli drive down TH17 cells and contribute to normal blood pressure

Most clinically diagnosed hypertensions, high blood pressure, are idiopathic in origin, meaning one can't determine what causes it. First thing patients are asked to do is to modify their diet habits and reduce salt intake. Diet high in salt thought to contribute to hypertension by water retention.

However, it could be that there is another, immunological pathway that contributes to high blood pressure. New study in Nature showed that in mice and maybe in humans as well, high salt diet depletes Lactobacilli, a gut microbiota species shown to lower TH17 differentiation and contributing in maintaining normal blood pressure.

Initially, the authors showed that in mice high salt diet (HSD) depleted several microbiota species, most notable, Lactobacillus murinus (initially identified through sensitive machine learning approach using the AdaBoost classifier from scikit-learn module run on Python).

In autoimmune model, adding Lactobacillus murinus could abolish high salt diet-induced increase in disease severity. Lactobacillus murinus appears to drive down TH17 differentiation (of note, high salt diet did not change TH17 population in germ-free mice).

Mechanistically, the authors showed that Lactobacillus murinus could inhibit TH17 differentiation by producing indole-3-lactic acid (ILA), a product of tryptophan metabolism.

Finally, volunteers on high salt diet display high TH17 differentiation and decrease in gut content for Lactobacilli.

In summary, the authors want to make the case that high salt diet could induce high blood pressure by depleting Lactobacilli and increasing TH17 cells which appear to initiate hypertension-related changes. 

It is clear that depletion of Lactobacilli per se is not sufficient for initiation of hypertension but we don't know what are other remaining microbiota species that actually induce TH17 in humans. Also, it is not clear whether microbiota-derived conserved molecules or metabolites (ILA) are sufficient for initiating hypertension or there are more complex events, such as chronic antigen-specific interactions that are ultimately responsible for sustaining chronicity of TH17 response and hypertension.    

posted by David Usharauli 

Infection-induced cell apoptosis activates self-epitope reactive T cells but barely

This week journal Nature Immunology published a study that had great title but poor data and conclusions. In fact, I rarely read such weak study for a long time, especially from top subject-matter journal. 

Basically, the authors tried to show that infection-induced cell apoptosis leads to self-peptide presentation and self-reactivity or even autoimmunity. Data are however misleading.

For this study the authors used the rodent pathogen Citrobacter rodentium that infects intestinal epithelial cells and induces their apoptosis. As a control, they have used infection with ΔEspF Citrobacter rodentium, a variant that lacks the secreted protein EPEC that mediates apoptosis. Initially, they showed that infection with WT Citrobacter rodentium, but not ΔEspF Citrobacter rodentium, induces Th17 response from large intestinal lamina propria (LI LP).

Next, the authors tried to examine whether infection-induced apoptotic cells will also provide self peptides for T cell activation (alongside of Citrobacter rodentium peptides). To do it, they have used so called double transgenic (DTg) mice derived from crossing OT-II mice with Act-mOVA mice. Now, these DTg mice delete absolute majority of OVA-specific OT-II cells in the thymus (from 1.5x10^6 to ~1,000 cells, i.e >1000X fold reduction of auto-reactive cells). The authors noted that DTg mice did not spontaneously develop autoimmunity and were healthy.

Next, when DTg mice were infected with Citrobacter rodentium, some portion of those OT-II cells left in DTg mice responded to it by up-regulating IL-17. The authors did not quantify the number of responding self-reactive OT-II cells and dot plot analysis reveals that their numbers seemed extremely low (on contour plot analysis). Moreover, it is not even clear whether self-reactive OT-II were responding to self-antigen or simply to inflammatory cytokine milieu [homeostatically] since even un-infected DTg mice showed proliferation and IL-17 expression in LI LP self-reactive OT-II cells.

The authors also showed that when infected with Citrobacter rodentium DTg mice showed little spike in anti-OVA IgA response driven by OT-II cells. However, it is not clear whether this anti-OVA IgA response has any pathogenic role.

Still, the authors believed that Th17 OT-II cells generated in DTg mice upon Citrobacter rodentium infection played pathogenic role in gut inflammation. As a "proof" they provided H&E staining of sections of large intestine from wild-type and DTg mice on day 40 after infection. Now, if scale bar on this H&E staining is 250 μm on both sections, then it is obvious DTg mice intestine is almost 2x more swollen or inflamed. But the authors noted that "DTg mice did not exhibit altered susceptibility to C. rodentium relative to that of wild-type or OT-II mice" and OT-II depletion did not significantly modify gut inflammation. So it is not clear from these data whether anti-OVA IgA or Th17 response after Citrobacter rodentium infection were in fact driving those observed pathogenic changes in the DTg mice guts (use of IL-17KO OT-II cells would have provided some guidance on this matter).

In summary, in my view this study only showed that WT Citrobacter rodentium infection induces little Th17 response from self-reactive T cells, however it failed to show that such Th17 response had any consequential effect.

David Usharauli  

New molecule, BHLHe40, links adjuvant activity of pertussis toxin to T cell pathogenicity in brain inflammation

Multiple sclerosis (MS) is a human neuro-inflammatory disease of autoimmune nature. Mouse model of MS is called experimental autoimmune encephalomyelitis (EAE) and it's induction in mice depends on dirty little secret: to induce EAE, mice are injected not just with peptide derived from myelin oligodendrocyte glycoprotein (MOG35-55) emulsified in CFA (to activate T cells) but mice are also injected with the co-adjuvant pertussis toxin (PTX), an ADP-ribosylating exotoxin derived from Bordetella pertussis (PTX is a whooping cough toxin that has been shown to be necessary for MS induction in this mouse model). No one really knows how or what way PTX primes mice for MS/EAE induction or why we even needed it in the first place (by the way, next time you hear that another drug failed in clinical trial for multiple sclerosis you know now it is because mouse model is completely artificial).

In this regard, new paper in Journal of Experinental Medicine is of interest. The authors report that transcription factor basic helix–loop–helix family member e40 (BHLHe40) was required for adjuvant activity of PTX for EAE development.

Using BHLHe40^GFP mice the authors first showed that CD4 T cells expressing GFP (surrogate marker for BHLHe40) were enriched for effector T cells expressing cytokines implicated in EAE pathology (IFN-γ, IL-17, GM-CSF).

Next, the authors showed that ADP-ribosylating activity of PTX was necessary to augment GFP/BHLHe40 expression in T cells and to induce EAE.

Adoptive transfer of BHLHe40-deficient 2D2 T cells (that are specific for MOG peptide) confirmed that BHLHe40 expression was required for T cell pathogenicity in EAE.

In summary, this study showed that during EAE induction PTX sensitizes both innate cells (for IL-1 production) and adaptive immune T cells (for GM-CSF and IFN-γ production) via action of BHLHe40.

David Usharauli

CD5-like likes to go easy with Th17

Two new studies about Th17 cells were published in journal Cell. Using single cell RNA-seq, these papers found additional molecular markers of Th17 pathogenicity. One of those new molecules was CD5L, a lipid scavenger receptor.

The authors, led by Vijay Kuchroo, found that CD5L expression segregated with non-pathogenic Th17 cells [induced in vitro with TGF-β1 and IL-6].

In addition, CD5L expression segregated with non-pathogenic Th17 cells in an in vivo brain inflammation model as well (compare CNS vs. spleen or intestine).

This observation was confirmed in CD5L-KO mice which showed exaggerated brain inflammation.

Adoptive transfer of in vitro [TGF-β1 and IL-6]-differentiated CD5L-KO Th17 cells indicated that absence of CD5L in T cells were sufficient to increase their pathogenicity in brain inflammation model.

Mechanistically, the authors showed that exaggerated brain inflammation with CD5L-KO T cells were related to increased production of IL-17 in Th17 effector stage [but not during naive T cell differentiation to Th17].

Since CD5L was previously implicated in lipid metabolism, the authors tested whether Th17-specific CD5L expression affected their cytokine profile via lipids. Indeed, this was a case. Specifically, it appeared that CD5L was antagonizing RORγt activity and its sensitivity to endogenous lipid ligands [such as oxysterols, a cholesterol metabolites].

In addition to CD5L, second paper in Cell showed Gpr65, Plzp, Toso were individually contributing to Th17 pathogenicity.  

In summary, involvement of cholesterol metabolites in Th17 pathogenicity is clinically highly relevant.

David Usharauli

Two distinct, mutually exclusive immune signatures, TH2 and TH17, determine therapy responsiveness in asthma

Asthma is a chronic condition manifested in episodes of airway hypersensitivity (inflammation, smooth muscle constriction) to innocuous signals that ordinarily would cause no tissue response in average individual. Eventually this chronic state leads to tissue remodeling and reduction of oxygen-rich air available for lung. 

Classical, allergic asthma episodes are driven by type 2 (TH2) immune response dominated by IL-4 and IL-13. However, more recently another category of asthma characterized by TH17 signature has been described. It appears that TH17 signature asthma may be more resistant to current asthma therapy.

The new study in Science Translational Medicine provided additional support for asthma stratification based on immune signature. This Genentech study showed that asthma immune profile can be stratified into mutually exclusive, non-overlapping TH2^high and TH17^high signatures.

This is a short paper. Basically, the authors analyzed airway gene signature from 51 asthma patients. They found that asthma patients' immune signature fall in 2 categories: either TH2^high or TH17^high. There is additional double negative category but its immune signature is not known.

Interestingly, both TH2^high and TH17^high signatures were associated with eosinophil infiltration.

Since current asthma therapies mainly target TH2 signature, the authors tested the outcome of α-IL-4/IL-13 blockade on animal asthma model (house dust mite antigen sensitization). As expected, dual blockade of TH2 cytokines significantly reduced airway inflammation.

However, TH2 cytokine blockade also induced TH17 signature, as would have been expected from in vitro studies (though it is not entirely clear from the data if this shift to TH17 pathway induced any clinically-relevant airway inflammation here).

Finally, the authors showed that blockade of both TH2 and TH17 pathways may be necessary to avoid inverse increase in TH2-driven airway hypersensitivity during anti-IL-17 therapy.

In summary, this study suggests that clinical trial design for α-IL-17 target therapy in asthma patients may need modification based on this findings. For example, α-IL-17 therapy alone may be not sufficient or that eosinophil signature could not be used as a exclusion factor for α-IL-17 therapy.

David Usharauli

TH17-derived IL-26 punches holes in bacteria

T_H17 cells have been implicated in host's protection against extracellular bacteria. It is thought that T_H17 cells recruit neutrophils and other granulocytes to the site of bacterial infection who do the job.

However, a new study in Nature Immunology provided evidence that human T_H17 cells can directly eliminate extracellular bacteria via release of IL-26, a cationic peptide that punches holes in bacterial membrane.  

This is actually very well done study. Initially the authors showed that recombinant human IL-26 (rhIL-26) had a direct bactericidal activity in vitro.

Next, the authors showed that rhIL-26 had bactericidal activity in vivo as well.

The authors found that in humans IL-26 expression was restricted to T cells and that out of T cells, T_H17 cells expressed high level of IL-26 and that T_H17 cell supernatant showed IL-26 dependent bactericidal activity.

In addition to its direct bactericidal activity, rhIL-26 could activate human plasmacytoid DCs (pDCs) when combined with dying bacterial DNA.

Interestingly, the authors showed that rhIL-26 could also combine with human DNA and activate pDCs and to a lesser extent monocytes.

Further experiments revealed that rhIL-26 + human DNA conjugate were taken up by pDCs and could signal through TLR9.

Finally, the authors showed that T_H17 cells could activate human pDCs to produce IFN-α via IL-26 + DNA conjugate formation.

In summary, these results provided mechanistic model for direct bactericidal activity of T_H17 cells against extracellular bacteria. In addition, results with IL-26 + hDNA conjugates and pDCs activation revealed how T_H17 cells could be involved in amplification of immune response during autoimmune conditions.    

David Usharauli

exTh17 cells cannot go unnoticed

Interleukin-17 (IL-17) is clinically relevant target molecule. T helper subset called Th17 are thought to produce large amounts of IL-17. Paradoxically, IL-17 is implicated in several human diseases such as psoriasis and inflammatory bowel disease (IBD), as well as autoimmune arthritis and autoimmune encephalitis. So, scientists are naturally very curious to understand Th17 subset's biology.

This new paper in journal Nature provided half-hearted data suggesting that Th17 cells undergo trans-differentiation into "regulatory T cells".    

Initially when I saw the article's title and read its abstract I thought it was about Th17-Foxp3 conversion since the authors used such term such as "regulatory T cells" which ordinarily refers to Foxp3+ CD4 T cells. However, it turned out results had nothing to do with Foxp3 T cells. 

Initially, the authors showed that in both steady state or during α-CD3 induced inflammation, CD4 T cells expressing IL-17 could lose its expression and instead up-regulate IL-10 (called here Tr1^exTh17 cells). However, for some reason the authors failed to show IL-10 expression level on CD4 T cells maintaining IL-17 expression (this could have been useful control). In addition, the authors did not discuss or show whether presence of two different types of IL-17A gene in a single cell could affect their individual expression maximum.

Next, the authors showed that Tr1^exTh17 cells could prevent colitis development in RAG KO hosts when co-transferred with pathogenic Th17 cells.

The authors also showed that anti-CD3 injection in mice immunized 35 days earlier with brain protein MOG  could induce Tr1^exTh17 cells.

These data so far suggest that non-physiological stimulation of T cells with α-CD3 antibody induced down-regulation of IL-17 and up-regulation of IL-10 in a subset of activated CD4 T cells. Whether these Tr1^exTh17 cells are derived from bona fide Th17 is not clear.   

Next, the authors used two disease models.  In Th2 dominant disease model of Nippostrongylus Brasiliensis, the authors observed the development of Tr1^exTh17 only after 2nd round of re-infection.

In a second Th17 dominant disease model of Staphylococcus Aureus, however, the authors conclusions that there is Tr1^exTh17 cell development is premature, in my view. The authors simply did not apply proper flow cytometry gating in this case.

In summary, in my view the correct interpretation of the data in this paper is following:

1.  Tr1^exTh17 can develop from activated T cells previously expressing IL-17, however those cells may not be bona fide Th17 cells.  

2. Effect of non-physiological stimuli such as α-CD3 antibody on T cells differentiation may lead to incorrect conclusions.

3. Based on results, in Staphylococcus Aureus model, there is no or very limited trans-differentiation of exIL-17 expressing T cells into Tr1^exTh17.   

Shortly, the major flaw in this paper is its heavy focus on gene-modified mice (on technology) rather than on biology.    

David Usharauli