Signal 1 = Signal 2 = Signal 3?

Occasionally one finds research articles published in the top journals, which rather than simplifying and clarifying the subject matters, make them more confusing. Frequently, even after reading and then re-reading them, one still cannot comprehend the "significance" of the finding.

I have one rule: if I cannot understand an immunology research paper with one read, I blame the editors. The author, of course, wants to publish his/her research in top journal, but it is a responsibility of the editors to be a gatekeepers and to make sure that the article is written in an easy to understand style.

I have another rule: the good data naturally make paper writing and reading easy.

The following paper from journal Science is such a disappointment. It starts strong with FucciRG mice model to track T cells acquiring a quiescent state after active cycling. The rest is confusing. The authors proposed that signal 1 (antigen), signal 2 (co-stimulation), and signal 3 (inflammatory milieu) play equal and quantitatively linear role in imprinting T cell division rate (they called it division destiny (DD), as if it would make it easier to understand). 

First of all, if one takes, for example cytokine IL-2, it not just quantitatively affects the T cell division rate but it qualitatively changes T cell fate and memory potential too. The authors suggestion that IL-2's physiological role is to maintain T cell division at a later stage of activation, at the tissue site, away from priming site (such as spleen or lymph nodes) does not account for IL-2's role in T cell effector class differentiation (CTL, IFN-gamma or IL-2 producers) or memory imprinting. These latter concepts could equally well explain the authors' observation with IL-2 receptor alpha deficient T cells.

I personally would suggest to Science editors to avoid publishing research papers filled with unnecessary models (especially mathematical models) or with one-sided explanations.

David Usharauli

Complement C3's inside job

I was very excited when I saw first time a research article about novel function of complement C3. It was published in September 5 issue of journal Science and as the title suggested it showed as C3 having intracellular signaling capacity.

But after reading it, I am a little bit confused. Data presented in this paper do not necessarily support the authors conclusions, at least unequivocally. Surprisingly, the results in this paper are generated fully from the in vitro studies, that is by itself very rare, almost unheard of, for a Science paper with the focus on immunology. 

Moreover, if one considers an in vitro experiment, one expects to see an experimental design that incorporates specific gene knockout (KO) cells, minimum. However, not a single KO cells are employed. For specific gene inhibition or specific protein depletion, the authors have used either (a) siRNA or (b) chemical inhibitors or (3) serum depleted of specific factors or antibody. However, none of these treatments provide the "clean" results. 

Still, few results are worth mentioning, though with limitations discussed above.

First, using NF-kB-driven luciferase reporter HEK293T cell line, the authors showed that incubation of cells with adenovirus type 5 vector (AdV) in presence of serum (but not AdV alone) could activate NF-kB. Activation of NF-kB was equally diminished when serum was either (a) heat inactivated or (b) antibody depleted. However, activation of NF-kB was completely abolished when heat inactivated antibody depleted serum was used. The authors speculated that both antibodies and complement have unique functions in NF-kB activation when exposed to AdV.

Next, the authors showed that transfection of cells with beads incubated with C3fBfD (alternative complement activation), but not with beads or C3fBfD alone, could activate NF-kB.

Interestingly, using AdV-GFP construct, the authors showed that AdV+serum combination generated factors that inhibited AdV replication.

Mechanistically, using siRNA technology, the authors speculated that serum complement effect was mediated via MAVS (RNA recognizing intracellular molecule). However, the authors did not show whether C3 could interact directly with MAVS (for example, co-immunoprecipitation).

In summary, this paper tries to suggest that C3 attached to viruses or bacteria are internalized by the cells and signal through MAVS to activate NF-kB and produce cell autonomous defense  state.

David Usharauli

    

Copying natural mutations to study autoimmunity

Autoimmune disease is a condition when body's immune system attacks its own tissue. Very little is known how autoimmunity is initiated or maintained. Frequently, we do not even know what are the (self)-antigens that are targeted by self-reactive T cells or auto-antibodies.  

The new paper in journal Science describes an one way how to identify the antigens targeted in autoimmunity. This study, led by Shimon Sakagushi at the Institute for Frontier Medical Sciences, Kyoto University, used SKG mice that has natural mutation in a T cell receptor signaling molecule, called ZAP-70. This mutation alters T cell selection in the thymus and allows peripheral release of self-specific T cells.

Original studies on SKG mice showed that they spontaneously develop arthritis mimicking several hallmarks of human rheumatoid arthritis (RA).  

Here, initial experiments showed that SKG T cells expressing different Vbeta chains could induce arthritis equally well when transferred into RAG-KO hosts, implying the poly-clonal nature of self-reactive T cells in SKG mice.   

The authors focused on one particular TCR family, Valpha2/Vbeta6 that constitute ~ 1% of joint infiltrating T cells. They cloned several Valpha2/Vbeta6 TCR pairs obtained from SKG arthritic joints, transfected them into RAG2-KO/SKG BM cells and transferred these transfected BM cells into RAG2-KO to generate monoclonal, retrogenic mice.

Out of these retrogenic SKG mice, R7-39, but not R1-23, spontaneously developed arthritis and skin inflammation. As expected, 7-39 TCR on wild-type background, W7-39, (without ZAP70 mutation) did not carry arthritogenic potential, probably due to inactivation in the thymus (and neither 7-39 nor 1-23 TCR develop into Foxp3+ T cells).

To examine the specificity of antigen(s) recognized by R7-39 T cells, the authors co-transferred 7-39 TCR transfected RAG2KO-SKG BM cells together with TCRbeta KO BM cells into RAG-KO hosts. In this setting, B cells developed from TCRbeta KO BM cells will secrete antibody specific to antigens recognized by 7-39 TCR, since only this type of help is available to B cells. Indeed, sera obtained from this mice, "R7-39+B", reacted to protein, RPL23A, identified by mass spectrometric analysis as a component of 60S subunit of ribosomes.

As expected, T cells from R7-39 mice, but not from R1-23, produced inflammatory cytokines in response to recombinant RPL23A.

Finally, the authors showed that sera from RA patients too showed reactivity to RPL23A.

In summary, these results shows one way to study mechanisms of autoimmunity. 

Two questions come to mind: it is very strange that SKG mice develop only limited set of autoimmune diseases (joint and skin inflammations), since ZAP70 signaling should affect every T cells. Why is that? Also, RPL23A is a widely expressed protein. So why are target tissues limited to joints and skin in SKG mice? Does it mean that level of expression determines clinical signs? or maybe microbiota play a role?

David Usharauli

What is high fat diet (HFD) good for?

It appears that even high fat diet, HDF, a modern day culinary and medical villain, has a positive characteristics, at least one we know of.  

This is what a new paper in journal Nature suggests. This research, led by Thirumala-Devi Kanneganti at St. Jude Children's Research Hospital, Memphis, studied mice with Pstpip2 mutation that spontaneously develop bone inflammation and bone erosion.

Surprisingly, the authors reported that these mutant mice when fed with high fat diet, but not regular low fat diet, showed no sign of bone inflammation.

The authors found that there was a correlation between the disease progression and abundance of gut flora microbe Prevotella.   

Interestingly, high fat diet could reduce the abundance of Prevotella in Pstpip2 mutant mice, implying that diet-induced alteration in gut flora contributed to disease protection.

Indeed, antibiotic treatment of Pstpip2 mutant mice could dramatically reduce disease development.

Alternatively, transplantation of fecal microbiota from HFD-fed Pstpip2 mice, but not low fat diet-fed Pstpip2 mice, into young Pstpip2 mice could prevent development of bone inflammation.

Mechanistically, HFD beneficial effect on bone inflammation was related to the reduction of IL-1beta production in HFD-fed Pstpip2 mice. 

Finally, the authors showed that antibody depletion of neutrophils could prevent bone erosion and inflammation in Pstpip2 mice.

In summary, the authors proposed that diet-induced gut flora modification alters IL-1beta mediated inflammatory bone damage in Pstpip2 mice.

HFD is usually shown to have a negative effect on metabolic syndrome, on development of type II diabetes, for example. The results in this paper are exception to this rule. Interestingly, Pstpip2 mice did not gain weight on HFD diet, implying that thermo-regulation and energy expenditure is differently regulated in Pstpip2 mice. Is there any role of innate lymphoid cell type 2, ILC2? 

David Usharauli    

  

Reverse engineering the T-independent type 2 (TI-2) antibody responses

A very interesting and thought-provoking research in humoral immunity was published in journal Science this week. It came from Bruce Beutler's lab at the UT Southwestern Medical Center. Professor Beutler, as many may know, is a 2011 recipient of Nobel Prize in Physiology or Medicine for his discovery of endotoxin receptor (TLR4).

Actually, before I review this paper I would like to highlight that since 2005 when Ruslan Medzhitov's lab has published the first paper in Nature that suggested the role of TLR in B cell responses and then later in 2006, David Nemazee's lab has published new study in Science questioning the validity of 2005 paper, there was a controversy in this field. In fact, in 2009, Ruslan Medzhitov's lab has published another study that confirmed that haptenated proteins used in 2006 study could promote antibody response independent of TLR signaling. However the mechanism remained elusive.

It appears that this new study in Science may be a first glimpse in the darkness to reconcile the differences.

I also would like to point out that Professor Beutler's lab is famous for studying ENU-induced random mutations in mice to generate unbiased molecular signature of immune deficiencies.

Humoral immunity is divided into T-dependent and T-independent (TI) antibody response. TI responses, are in turn segregated into two categories: TI-1 and TI-2. This new study focus on TI-2 response.

Initially, using NP-Ficoll immunization, as a TI-2 model antigen, the authors observed that NP-specific IgM response was diminished in single STING-KO, cGAS-KO, MAVS-KO mice but not in several TLR signaling KO mice. 

Mice, double deficient in cGAS/MAVS, had almost complete absence of NP-specific IgM response.

This was strange observation since STING, cGAS and MAVS are known to detect the presence of DNAs or RNAs and NP-Ficoll contained none of them.

Using adoptive B cell transfer from these three KO mice into RAG-KO hosts, the authors showed that this effect of NP-Ficoll was B cell intrinsic (and the number of B1 or MZ B cells were normal in non-responding KOs).

The authors concluded that DNAs and RNAs were generated as a result of B cell stimulation by NP-Ficoll. Indeed, several endogenous retroviruses were up-regulated in NP+ B cells as compared to NP- B cells.

Furthermore, NP-ficoll immunization induced Reverse Transcriptase (RT) activity in NP+ B cells.

Parallel experiments revealed that mice deficient in NF-kB signaling also lacked the ability to respond to NP-Ficoll immunization or express endogenous retroviruses.

The authors observed that B cells from MAVS-KO, but not from cGAS KO mice, showed reduced phosphorylation of NF-kB proteins after anti-IgM stimulation, implying that MAVS played more critical role in sustained activation of NF-kB.

In summary, the authors proposed the new model of TI-2 antibody response. According to this model, when antigens with repetitive epitopes engage specific B cells, this activates initial wave of NF-kB activity leading to expression of endogenous retroviruses as a RNA that are subsequently converted into cDNA by RT activity. These newly generated RNAs and cDNAs are recognized by MAVS and cGAS/STING pathways, respectively. In turn, MAVS activation induces second wave of NF-kB activation to sustain TI-2 antibody response.

As the authors correctly pointed out, at this stage, it is not clear whether expression of endogenous retroviruses in activated B cells indeed play a critical role in TI-2 response. Their presence could be coincidental to this process. It would require B cell devoid of endogenous retroviruses to definitely test their precise role (since both TI-1 and T-dependent antibody response were shown to be STING, MAVS and cGAS independent).

For me this results represent new concept how we should assess the role of endogenous viruses present among our genes. Not everyone carries them, even among laboratory mice strains there is significant differences. Why would nature develop such system where TI-2 response would depend on endogenous viruses? It is just a fascinating idea.  

David Usharauli

Friends or Frenemies?

This is a second paper from Host Cell and Microbe describing how mouse gut residing commensal microbe's metabolic end-product, succinate, fuels the virulence of pathogenic Citrobacter rodentium (C. rodentium).

This study, led by Vanessa Sperandio at UT Southwestern Medical Center, Dallas, analysed virulence factor expression in mouse C. rodentium, in presence of mouse gut commensal Bacteroides thetaiotaomicron (Bt). The authors have used C. rodentium disease model that mimics E. coli infection in humans.

It appears that the authors' initial objectives were to study the mechanisms by which gut commensals were driving virulence factor expression in pathogenic microbes and only later diverted their attention towards the role of succinate metabolism in this process.

First, the authors showed that presence of Bt enhances expression of several virulence factors in E. coli and in an in vitro culture.  

Similar results were observed with C. rodentium  culture.

Interestingly, this enhancement was not observed in cra mutant E. coli that is incapable of sensing sugar fermentation products, like succinate (however, the authors did not show similar results with cra mutant C. rodentium).

In vivo experiments with either wild-type C. rodentium or mutant strains showed that reconstitution of antibiotic treated mice with Bt could enhance C. rodentium pathogenicity. The authors reported that severity of infection with cra mutant C. rodentium  was attenuated, though not abolished, it appears (implying that C. rodentium showed cra-succinate independent virulence).

Still, analysis of sugar fermentation products in the day 2 post infected mice cecum showed that there was selective increase in succinate in presence of Bt or C. rodentium.

Finally, addition of succinate to E. coli culture could enhance expression of virulence factors in the wild-type but not in a cra mutant E. coli strain (again, the authors did not show similar results with cra mutant C. rodentium).

In summary, this study and another one from Justin Sonnenburg's lab, highlighted the potential detrimental role of gut commensals in fueling the virulence of pathogenic microbes. We are thinking that the term gut commensal implies friendly microbe, however occasionally those friendly microbes could involuntarily provide support to pathogenic ones.

David Usharauli

Fueling C. difficile growth

C. difficile is a spore-forming anaerobe, residing in the human gut. Usually it does not cause any pathology. However, antibiotic use (or over-use), or changes in gut motility can bring up the ugly side of this microbe. There are no vaccines or drugs available for the specific management of C. difficili infection. 

Curiously, in the past few years, several research studies indicated that C. difficile could be controlled by other gut microbes readily available in the healthy humans' feces.

If proven effective in humans, this will open a new chapter in medicine. Our physiology is heavily influenced by gut commensals and their role in human health and disease has just started to be unraveled.

In this respect, two new studies in journal Cell Host and Microbe provided additional clues about potential mechanisms of initiation of C. difficile disease. I will review both of them separately.

One study, led by Justin Sonnenburg at Stanford University of School of Medicine, has examined how fermentation end product, succinate, affect C. difficile growth pattern.

Initially, the authors observed that in germ-free mouse, C. difficile growth was accelerated in the presence of polysaccharide rich-diet and mouse gut flora commensal Bacteriodes Thetaiotaomicron, (Bt).

It is known Bt produces organic acid, succinate, as a fermentation end product. 

Using succinate-transporter mutant C. difficile, the authors confirmed that in vitro culture, WT C. difficile were able to utilize excess succinate for their growth enhancement.

In vivo experiments with succinate supplemented diet validated this observation.

Importantly, the authors showed that Bt-induced growth promotion was not observed with mutant C. difficile.  

To make their observation clinically relevant, the authors conducted the series of experiments with mice treated with antibiotics or gut hyper-motility (diarrhetic) drug.

As predicted, antibiotic treatment specifically increased succinate level.

Similarly, diarrhetic agent mimicked antibiotic's effect.

Finally, the authors showed that changes in gut motility fueled C. difficile growth.  

In summary, these results indicate that changes in gut flora or acceleration of gut motility could lead to specific increase in succinate level that is sensed and utilized by C. difficile for growth enhancement.

David Usharauli

Stress-release with IL-22

Type II Diabetes (T2D) is part of a complex metabolic syndrome. Typically, conditional insulin deficiency as observed during T2D is a result of an imbalance between production and demand for insulin. I always wondered why would nature develop such a system where packaging of excess glucose would be controlled by a single molecule (insulin), while glucose unpacking, it's release into bloodstream are controlled by so many molecules, including corticosteroids.

Prevalence of T2D is increasing in modern population. Speculation varies as to the cause of such increase in T2D (availability of excess food, poor physical activity, widespread use of broad-spectrum antibiotics causing changes in gut microflora, etc).

Frequently the metabolic syndrome is associated with the changes in immune cytokines. Occasionally, immune cytokines are directly implicated in disease, for example IL-17 and TNF-alpha in psoriasis or rheumatoid arthritis.

On other hand, the list of immune cytokines are constantly expanding and maybe some of them would show a beneficial effect.

This new paper in Nature Medicine is one such research. This study, led by Michael McGuckin from the University of Queensland (Australia), has uncovered the protective role for cytokine IL-22 in T2D. 

IL-22 is a member of IL-10 cytokine super-family that includes IL-10, IL-22, IL-24, IL-26, so far.

Using in vitro assay to measure beta cell ER stress, the authors showed that IL-23, IL-24, IL-33 were the potent inducers of ER stress.

Parallel experiments with beta cell exposure to the pairwise combination of cytokines revealed that IL-22 (and IL-10) could reverse ER stress caused by any other cytokines examined, including IL-23, IL-24, IL-33. Two exceptions were IL-17A for IL-22 and IL-17F for IL-10 for which the effectiveness of such inhibition were less prominent.

As a consequence of reduced ER stress, IL-22 was able to improve secretion of insulin from primary mouse beta islets.

Importantly, using high-fat diet induced obesity mouse model (HF-DIO mouse), the authors showed that in vivo treatment with IL-22 could reduce beta cell ER stress and reverse decline in insulin secretion in response to HF diet.

In addition, IL-22 treatment could reverse high-fat diet effect on body weight and glucose tolerance.  

Mechanistically, this beneficial effect of IL-22 was associated with decrease in ER-stressor inflammatory cytokines and in increase for enzymes responsible for neutralization of free radicals.

Finally, the authors showed that this beneficial effect of IL-22 could be reproduced in an in vitro experiments with human beta islets as well.

In summary, this figure-packed research showed that IL-22 can have a protective characteristics beneficial for management of beta cell functions.

It is not immediately clear why immune cytokines play such important roles in non-immune functions. For example, why would IL-23 induce beta cell stress and reduce insulin secretion? Similarly, it is not clear why IL-22 would develop protective function for beta cells. Previous studies showed that IL-22 has a protective role in gut epithelial cell function (probably through it's effects on cell surface fucosylation). On the other hand, IL-22 has been implicated in promoting skin epithelial hyper-proliferation characteristic to psoriasis. So, the field is quite confusing, to say the least.

David Usharauli

Commensal virus to the rescue

We all heard about gut microbiome and how an important job they do to keep us healthy. Still, there is the situations when the antibiotic therapy is necessary. 

Since many beneficial microbes in our gut are sensitive to broad spectrum antibiotics used in today's medicine, such treatment could lead to dysbacteriosis and gut inflammation. Now, beneficial microbiome therapy (fecal transplantation) is an obvious option, but such therapy could be inefficient due to the fact that such flora itself is sensitive to antibiotics. So what is the solution?

It appears that some friendly viruses in the gut could provide such beneficial, cover effect. Such study, led by Ken Cadwell from New York University School of Medicine, was recently published in journal Nature.

The authors studied the murine norovirus (MNV). This virus is endemic in laboratory mice colonies but does not produce an overt gut inflammation in a immune-competent host. 

Reconstitution of germ-free mice with MNV produced changes in the gut morphology and immune cells resembling conventional mice gut. 

More relevant, MNV could reverse antibiotic-induced negative effects on gut immune system.

Mechanistically, such beneficial effect of MNV on gut immune system was type I IFN dependent.

Finally, MNV could prevent antibiotic-induced gut sensitivity to chemical damage (DSS).

In summary, the authors showed that viral therapy could be an viable alternative (since virus would not be sensitive to antibiotic itself) to treat or prevent antibiotic-induced gut immune malfunctions.

Of course, application of this study to human population would require additional research. 

First, human Norovirus is clearly not a benign virus (cruise ship virus). So we need to find the virus that does not induce an overt gut inflammation in healthy humans. 

Second, this study examined immune-competent mice. However, analyses of immune-deficient mice responses to NMV would be more informative since in humans, antibiotic therapies are frequently used to compensate for immune deficiencies.

Third, if gut commensal, beneficial viruses exist, they should naturally (automatically) take over the beneficial microbes functions during antibiotic treatment. Do they do it?

Fourth, recent studies indicated that viruses, including MNV, require the presence of bacteria for their infectivity. How this knowledge affects the results of this study is to be determined.

David Usharauli

Tolerance in humans is a full-time work: CTLA4 haploinsufficiency

CTLA4 is a powerful inhibitory molecule required to keep activated T cells in check. For example, mice deficient in CTLA-4 develop fatal multi-organ immune inflammation, however mice with CTLA4 haploinsufficiency (where only one copy of the gene is defective) showed no apparent clinical signs.

This paper in journal Science describes an identification of 4 different families carrying one defective copy of CTLA-4 gene and (in contrast to mice model) showing immune related inflammatory disorder in multiple organs.

The authors observed that these individuals have reduced number of T regulatory cells with reduced capacity to inhibit T cells in a in vitro assay.

Next, it was found that T regulatory cells from these individuals expressed half the amount of normal CTLA4 level (though the authors failed to compare T regs' functionality sorted based on CTLA4 expression level: high, medium and low).

Additional in vitro experiments showed that transfection of patient's PBMC with healthy, wild-type copy of CTLA4 can reduce T cell proliferative response. 

Conversely, similar to patient's T cells, knock-down of CTLA4 expression by siRNA increased healthy PBMC proliferation (though in both these assays, measuring proliferation by simple gating on dividing population does not provide the full picture of CTLA4's effect).

The authors also observed that these individuals had reduced number of peripheral B cells. 

Finally, in contrast to T cells, in vitro proliferation assay for B cells showed dramatic reduction of proliferation from patient's B cells, probably due to fact that patient's PBMC lacked majority of memory B cells.

In summary, this paper identify the individuals with natural mutations in one copy of CTLA4 gene and having autoimmune phenotype. This indicates that in humans CTLA4 functions is a dose-dependent manner and requires presence of both two healthy alleles. Similar observation was reported by another research group in Nature Medicine (CTLA-4 haploinsufficiency results in disrupted T and B cell homeostasis). In general, results from these studies reminds data from mouse Foxp3 studies where T regs function were shown to be dependent on the level of Foxp3 expression.  

David Usharauli