α-IL23, not α-IL17 antibody blockade, shows protection in IBD model

Previous studies indicated that cytokines IL-23 and IL-17 act in concert to induce and perpetuate autoimmune inflammation. This is certainly true for psoriatic skin inflammation.

However, two new back-to-back studies in journal Immunity provided evidence that when it comes to intestine inflammation, role of IL-23 and IL-17 goes in opposite direction. Here, blockade of IL-17 exacerbate while blockade of IL-23 inhibits intestine pathology and gut wall permeability.

Both studies came from research groups working in biotech/biopharma (Daniel Cua's group at Merck Research Laboratories and Jennifer Towne's group at Amgen [presently at Janssen]). Both studies reached similar conclusions. 

Here, the authors treated colitis-prone mice with blocking antibodies specific for IL-23 or IL-17 cytokine family. Surprisingly, both α-IL17A or α-IL17RA treatment worsened gut pathology, while α-IL23 (p40 or p19 subunits) antibody showed protection.

Unlike α-IL23, α-IL17RA antibody treatment was associated with increased gut wall permeability (serum sCD14 and LBP), implying IL-17 role in gut health.

Similar increased gut leakage (with FITC-dextran) was observed by Merck's team in chemical irritant DSS-induced GI inflammation model in IL-17KO mice (though Amgen's team did not observe it in DSS mouse model with α-IL17 treatment. It could be that α-IL17 antibody blockade did not fully inhibit IL-17 action as it could be expected in IL-17KO mice).

Additional experiments showed that γδ T cells were the main producers of gut IL-17 in DSS model and that γδ T cell KO mice showed the same increased GI tract permeability as IL-17KO mice.

Finally, Merck's team showed that (a) γδ T cell-derived IL-17 production in the gut were mostly IL-23 independent and (b) IL-23rKO mice were protected against worsening GI wall pathology.

In overall, these two studies suggest that local gut tissue associated IL-23 independent but γδ T cell-derived IL-17 production plays a protective role in gut permeability. Basically, this means that α-IL23 blockade, but not α-IL17 pathway inhibition, would most likely provide benefits to patients suffering from GI tract idiopathic inflammation.

David Usharauli

Sialic acid decorated multi-valent nanoparticles prevent sepsis mortality in mice

Sepsis is an uncontrolled tissue-immune [hyper]response that leads to the organ failure and frequently patients' death. At present, there is no specific treatment for sepsis, just supporting care. Obviously, sepsis represents important unmet medical condition.

New study in Science Translational Medicine provided evidence that sialic acid coated nanoparticles could prevent sepsis-induced death in mice model of acute sepsis.  

This study is based on prior evidence that binding of sialic acid (type of sugar) to its receptors called Siglecs on macrophages or neutrophils could inhibit inflammatory pathways. Here, the authors showed that unlike bi-valent anti-Siglec antibodies, administration of multi-valent sialic acid coated nanoparticles (in a form of α2,8 sacharide linkage), at both T= 0h or T= +2h, prevented endotoxin induced animal death (i.p. injection).

Similar protection was observed in anesthetized mice undergoing cecal ligation and puncture (CLP) procedure (here too, α2,8-NANO was administered i.p.).

Additionally, intra-lung administration of α2,8-NANO showed significant (but not as impressive) protection against lung inflammation (a secondary complication following cecal ligation and puncture procedure).

The authors showed that protection provided by α2,8-NANO was IL-10 dependent in endotoxin injection model (though, for some reason, the authors had used 1mg α2,8-NANO here, not 2mg α2,8-NANO, as in other figures). Also, application of exogenous IL-10 in IL-10KO mice would have provided more clear role of IL-10 here. 

Finally, the authors tried to show that α2,8-NANO application could be useful for human condition as well. They showed that α2,8-NANO could reduce inflammatory cytokine secretion from primary monocytes stimulated with endotoxin in vitro (but for some reason the authors decided to show "processed", not raw data, implying that data were either not consistent or showed too much variability).

In summary, this study suggests that sialic acid decorated multi-valent nanoparticles may play beneficial role in sepsis management (I want to note here that I am against conducting such distressing experiments on lab animals as are i.p. injection of endotoxin or CLP. We must find better alternative models).

As mention earlier, this study comes with 2 major drawbacks (that make this study not easily "translational"):

(1) while animal studies, shown here, were quite impressive, primary human cells showed less sensitivity towards α2,8-NANO application.
(2) even in animal models, α2,8-NANO was applied through i.p. injection. This route of injection is completely non-applicable for human use and I am puzzled that labs still continuing using it. Why not just use injection via i.v. route?

David Usharauli

Species-specific microbial attachment to host epithelial cells determines gut IL-17 induction

Gut microbes drive maturation of host's immune system. Not every microbe is able to do it, however. So what microbial qualities determines its impact on host?  

New paper in journal Cell has revealed a simple rule of thumb: microbes that could attach themselves to gut epithelial cells drove induction of local Th17 cells and IgA. Interestingly this microbial quality found to be species-specific. 

Initially, the authors showed that monocolonization of germ-free mice or rats with endogenous segmented filamentous bacteria  (mouse-SFB and rat-SFB) induced Th17 cells in a species-specific manner (mouse data are shown here ).   

Ex vivo stimulation of lamina propria cells with autoclaved fecal antigens (A/C) showed that IL-17 secretion and IgA production were correlated with species-specific access to SFB antigens in the gut.

Additional experiments with WT microbes or microbes lacking adhesion molecules confirmed that epithelial adhesion determined IL-17/IgA production.

In summary, these results indicate that direct adhesion to gut epithelial cells is necessary pre-condition for local immune response induction. Mechanistically, this adhesion by microbes induces production of serum amyloid A (SAA) that primed local T cells for Th17 phenotype.

David Usharauli

  

Cytosolic DNA sensor cGAS controls systemic autoimmunity to self-DNA

Type 1 IFN system (IFN-α, IFN-β, etc) is a complex and powerful immuno-regulatory circuit that show both anti-viral (or general pro-inflammatory) or anti-inflammatory activity. Similar to IL-12p70, type I IFNs were shown to prime naive T cells for anti-tumor activity. Very few cytokines have such direct priming effect on naive T cells. 

However, it was not clear how tumors were detected by IFN circuit. Later, cytosolic DNA sensor cGAS and its adaptor molecule STING were discovered and implicated in tumor DNA sensing and T cell priming via IFN circuit.

Unlike viral or bacterial DNA, self-DNA are not ordinarily accessible to cGAS in the cytoplasm. But in disease state when there is an excess of self-DNA, cytoplasmic cGAS could become activated and mistakenly initiate "anti-viral" cascade to self-DNA leading to debilitating immunopathology.

This is a story told by two recent papers, one published in Journal of Immunology and another in PNAS. In these papers, the authors showed that mice knockout for self-DNA degradation enzymes (Trex1^-/- or DNase-II^-/-) succumb to immunopathology, but could be rescued by absence of cGAS (or STING).     

Both papers showed very similar results so I will going to mix them in my analysis. In brief, the authors showed that immunopathology in mice deficient for Trex1 (an exonuclease that degrades cytosolic DNA) could be completely reversed by absence of DNA sensor cGAS.

Mechanistically, Trex1^-/-cGAS^-/- double knockout mice showed reduced level of auto-antibodies, comparable to WT mice (immunoblots against heart Ag are shown).

Similarly, peripheral immunopathology in [DNase-II^-/- mice] that lack another cytosolic DNA degradation enzyme DNase-II could be completely rescued by simultaneous absence of cGAS.

In summary, these two studies clearly support the hypothesis that abnormal sensing of endogenous self-DNA by cGAS is a major priming step leading to immunopathology in diseases such as systemic lupus erythematosus (SLE), Aicardi-Goutieres syndrome, familial chilblain lupus, retinal vasculopathy and cerebral leukodystrophy.

In general, I agree that modulation of cGAS-STING pathway will provide enormous benefits in both tumor therapy and systemic autoimmunity.

David Usharauli

Perforin deficiency in dendritic cells leads to overweight and metabolic syndrome

Dendritic cells (DCs) are both necessary for immunity and tolerance. While immunity part is more or less clear, DC's role in tolerance is not clear at all.

Here is another weird tolerogenic DC paper from Immunity. Here, the authors found that in BM chimera mice that lack perforin specifically in CD11^hi DCs develop a metabolic syndrome (obesity, insulin resistance and low glucose tolerance).

To tell you truth, this study should not be in Immunity. Journal of Immunology would have been better place. The problem is that the authors did not even bother to check any role of gut micirobiota in all these. But if you mention metabolic syndrome and did not check for gut microbiota, this is a cardinal sin for today's immunology.

In brief, the authors have generated mixed BM chimera mice wherein only DCs in mouse would be derived from BM cells lacking perforin (though having CD11c-driven Cre model would have been better alternative). Unexpectedly, the authors found that starting at 3 month these BM chimera mice become overweight.

The authors found that these mice with CD11c-specific perforin deficiency developed large adipose cells.

Finally, they showed that metabolic syndrome in these mice could be reversed by T cell depletion (but the authors have no data to explain why or how presence of T cells induces metabolic syndrome).

In summary, according to authors, this study suggests that when DCs lack perforin and are not able to eliminate T cells, this leads to accumulation of T cells that promote metabolic syndrome. What or how is not known. No words about gut microbiome.

David Usharauli  

Antigenic cartography of dengue virus types reveal extensive promiscuity

Dengue virus, DENV, belongs to a genus flavivirus. DENV are segregated into 4 serotypes: DENV1-4 (though, in 2013, one study suggested the existence of 5th serotype). Each DENV serotype induces serotype-specific immune protection but strangely can cross-enhance clinical severity of other DENV serotype-induced infection. 

New study in journal Science tried to decipher the mystery of DENV immune response. Using antigenic cartography derived from the large set of DENV isolates and immunofocus reduction neutralization assays the study revealed that proportion of DENV within serotype have greater antigenic resemblance to DENV of a different serotype than to some DENV of the same serotype.

First, the authors created DENV antigenic cartography from samples derived from African green monkey after viral challenge. Note that (1) DENV3-4 sera (open circles) are clustered more tightly to corresponding viruses, compared to DENV1-2 sera and (2) proportion of viral isolates (closed circles) are more close to virus from other serotypes than to its own.

Next, the authors created DENV antigenic cartography from healthy volunteers vaccinated with monovalent DENV vaccines or from samples of DENV natural infection in humans.  Please note that here too vaccination with DENV1-2 induced less tight map compared to DENV3-4 vaccination or natural infection. 

In summary, these DENV data revealed that segregation of DENV isolates into distinct serotypes may not adequately explain DENV immune response. It also showed that unlike natural infection, DENV vaccine produced less-focused immune response, especially for DENV1 and DENV2.

David Usharauli

CTLA-4 deletion during adulthood leads to a paradoxical resistance to autoimmune disease

CTLA-4 is a T cell inhibitory molecule that competes with CD28 for binding CD80/CD86 co-stimulatory molecules on dendritic cells. It is believed that CTLA-4 functions as a "brake" for T cell activation, a function also referred as a "checkpoint" inhibition. Total genetic deficiency of CTLA-4 or just on Foxp3+ regulatory T cells leads to early onset wasting syndrome and fatal autoimmune diseases. This what we knew up to now. 

However, a new study in Journal of Experimental Medicine, has put upside down the whole concept of "checkpoint" inhibition for CTLA-4. Using genetically-modified mouse models, the authors reported that total or Foxp3+ regulatory T cell-specific conditional deficiency of CTLA-4 in adult mice leads to protection rather than acceleration of autoimmune disease.   

First, the authors have generated mouse model that allowed conditional deletion of total or Foxp3+ Treg-specific CTLA4 (UBC^Cre/ERT2+ Ctla4^fl/fl and Foxp3^{eGFP/Cre/ERT2+} Ctla4^fl/fl).

Next, the authors treated mice with tamoxifen to activate Cre recombinase and delete CTLA4 and challenged mice with myelin self-peptide to induce experimental autoimmune encephalitis (EAE). Surprisingly and contrary to widely held belief, adult mice conditionally deficient for CTLA-4 were highly resistance for EAE induction.

Similar resistance for EAE induction was observed with mice with adult-onset CTLA4 deletion specifically on Foxp3+ regulatory T cells.

To rule out any off-target effect of CTLA-4 deletion, the authors transferred T cells from UBC^Cre/ERT2+ Ctla4^fl/fl into T-deficient hosts and then challenged with tamoxifen and myelin self-peptide. Here too, deletion of CTLA4 on mature T cells protected against EAE. Moreover, adoptive transfer of purified, naive Foxp3- T cells from UBC^Cre/ERT2+ Ctla4^fl/fl mice or transgenic 2D2 T cells specific for myelin self-peptide on UBC^Cre/ERT2+ Ctla4^fl/fl background were capable of inducing EAE, implying that T cells were not inherently incapable of inducing EAE but were actively suppressed by absence of CTLA-4 on Foxp3+ regulatory T cells.

Additional experiments revealed that CTLA-4 deletion in adulthood skewed T cells response towards IL-10 production in UBC^Cre/ERT2+ Ctla4^fl/fl mice.

Finally, the authors showed that CTLA-4 deletion during adulthood did not modify anti-tumor response against MC38 colon adenocarcinoma cells in UBC^Cre/ERT2+ Ctla4^fl/fl mice.

In summary, these results question the assumption that inhibition of CTLA-4 function during adulthood would lead to autoimmune disease. Based on these results, it is unclear how anti-CTLA4 antibody therapy provides benefits during tumor immunotherapy. It maybe the difference between mouse and human immune system or it could be that mechanism of action of anti-CTLA4 antibody is not a "checkpoint inhibition" after all.

David Usharauli

Gut microbiota controls clinical severity of sickle-cell disease via neutrophil "ageing"

Our knowledge of the role of gut microbiome in human health and disease is expanding on a daily basis.

For example, just few days ago journal Nature published a study where the authors showed that in mouse model of sickle-cell disease (Hba^-/- Hbb ^-/-) the presence gut microbiome influenced clinical severity via neutrophil "ageing".      

In sickle-cell disease, neutrophils expressing Mac-1 capture sickle red blood cells that leads to vaso-occlusion and tissue damage. It appears that "ageing", or mature neutrophils, defined as CD62L^low CXCR4^high population, express more Mac-1 and those neutrophils can capture more RBCs per cell basis (that contributes to development of sickle-cell disease symptoms). [the authors does not discuss the difference between "ageing", "senescence" and "mature" neutrophils].

Next, the authors showed that germ-free mice or antibiotic treated mice have reduced number of "ageing" neutrophils.

Using BM chimera, the authors found that absence of MyD88 adaptor molecule or TLR4 receptor also mimicked the effect of antibiotic treatment on neutrophil "ageing".

Finally, the authors found that antibiotic treatment of sickle-cell disease model mice reduced number of circulating "ageing" Mac-1 expressing neutrophils and showed reduction in tissue damage.

In summary, this study proposed a novel therapeutic path for treatment for sickle-cell disease by modulation of gut microbiota and neutrophil maturation ("ageing").

David Usharauli

Pain sensing neurons alert dendritic cells for presence of skin fungal parasites

IL-23/IL-17 axis plays important role in the host's defense against fungal parasites. Recent studies show that skin CD11b⁺ dendritic cells and skin γδ T cells contribute for anti-fungal protection.

New study in journal Immunity added yet another layer of protection to the skin. It showed that skin pain and thermal sensing TRPV1 neurons also sensing the presence of fungal parasite and alert local immune defense system.

Using mouse model of candida albicans fungal infection, the authors first re-confirmed that cytokine IL-17 showed anti-fungal activity.

Next, the authors showed that the source of this protective IL-17 were skin γδ T cells (and not conventional αβ T cells or non-conventional dendritic epidermal T cells (DETCs).

Next, the authors re-confirmed that IL-23 was upstream of IL-17 in host's protection against candida albicans fungal infection.

Next, the authors showed that skin langerhans cells (LCs) and Batf3 ⁺ CD103⁺ DCs (LC^Δ Batf3 ^Δ) were dispensable for anti-fungal protection.

However, the authors found that IL-23 derived from C-type lectin positive dermal DCs (using Mgl2-DTR + IL-23KO BM chimera) were necessary for this protection.

Afterwards, the authors found that ablation of skin nociceptors, TRPV1, decreased anti-fungal protection.

Finally, the authors found that upon detection of candida albicans, CGRP (calcitonin gene related peptide) secreted by TRPV1 neurons acted on Mgl2+ dermal DCs to induce IL-23 secretion that in turn induce protective IL-17 from local skin γδ T cells (strangely, however, Fig. 6H and 6I do not match the authors text in the results. Here, -DT+CGRPα samples should have significantly lower CFU compared to +DT+CGRPα samples, since DT depletes Mgl2+ DCs).

In summary, these results provide additional support for neuro-immune network affecting host's protection against infection. This does not mean that neurons are important for ultimate clearance of the pathogen. Here, the authors only showed day 3 of infection when there is a maximum burden of fungal pathogen.

However, there is also a broader implications. Mainly, how CNS can influence or even imitate local immune response and produce or augment skin inflammation such as in psoriasis, dermatitis, eczema, urticaria and skin allergies.  

David Usharauli

Tumor cell-intrinsic effects of α-PD1 therapy in absence of adaptive immune system

α-PD1 antibody immunotherapy shows significant benefits in cancer patients. In some settings, α-PD1 immunotherapy is even superior to α-CTLA4 immunotherapy. Since both α-PD1 and α-CTLA4 immunotherapy work as the immune checkpoint inhibitors, question then is what determines such selective advantage of α-PD1 immunotherapy?  

New paper in journal Cell may provide the clue. Here, the authors reported that α-PD1 antibody could delay cancer cell growth independent of its role on adaptive immune system.

First, using several complementary approaches (Flow Cytometry, RT-PCR, In Situ staining), the authors confirmed that melanoma cells (both primary and cell lines in both human and mouse) expressed PD1.

Next, using mouse melanoma transplantation model, the authors showed that melanoma growth correlated with the level of PD1 expression (wherein PD1^high tumors grow faster [PD1 over-expression (OE)], and PD1^low tumors grow slower [PD1 shRNA]) and this effect was independent of adaptive immune system (NSG mice, see below).

Using anti-PDL1 antibody blockade, the authors showed that tumor growth promoting effect of PD1 over-expression (OE) was PD-1 specific (and not off-target effect).

Similar, cancer cell-intrinsic effect of PD1 signaling were seen with human melanoma cells transplanted in NSG mice (NSG mouse lack adaptive immune system and has minimal NK cell activity).

To verify cancer cell-intrinsic effect of PD1 in unmodified cells, the authors transplanted mouse parental melanoma cells (B16-F10) into wild-type, PD1 KO or NSG mice. Here, α-PD1 antibody therapy could delay tumor growth in PD1 KO and NSG mice, but strangely not in wild-type mice (this goes against human clinical trials).

However, more importantly, α-PD1 antibody therapy could delay patient-derived melanoma cell growth in NSG mice (it appears that human melanoma cells are more sensitive to α-PD1 therapy compared to mouse melanoma cells).

In summary, these results suggest that some portion of anti-tumor effect seen with α-PD1 antibody therapy could be attributed to the cancer cell-intrinsic effects of PD1 signaling, independent of its role on adaptive or innate immune system (i.e. independent of its role as a checkpoint inhibitor).

David Usharauli 

Dirt exposure could prevent asthma development in mouse

Allergic or exaggerated response to mostly innocuous antigens is a Terra Incognita in our understanding of how immune system really works. First question that comes to mind is why is there even such molecule as IgE that causes nothing but allergic reaction when combined with antigen? A hypothesis proposed by immunologist, Ruslan Medzhitov, suggests that the evolutionary role of IgE was to defend the body against toxins. But IgG could do that too? 

Another hypothesis commonly referred as hygiene hypothesis explains that allergic responses are, in fact, out-of-control type II immune reactions (highly skewed Th2-IgE-IL-13 axis) due to absence of "normal" education of immune system by reduced exposure to infections or infectious materials (bacteria, dirt, dust, fungi, dander, etc.) during early life (that supposed to induce Th1-IFNγ axis balancing Th2 response).

The new paper in journal Science is another example for such immune "balancing" act in support of hygiene hypothesis. Here, the authors reported that pre-exposure to low dose of LPS (endotoxin, a part of gram-negative bacteria) could raise the threshold for lung airway epithelial sensitivity to house dust mite (HDM) extract via modulation of NF-kb activity via up-regulation of A20 (Ubiquitin-editing protein).  

First, the authors showed that compared to control, mice pre-exposure to LPS before HDM application were protected from developing features of airway hypersensitivity (the most important figure is 1D).

Since LPS pre-exposure induced A20 up-regulation in lung epithelial cells, the authors tested mice with lung epithelial specific deletion of A20 (tnfaip3 KO). Indeed, beneficial effect of LPS exposure was abolished in these A20 deficient mice (tnfaip3 ^EC-KO).

Finally, the authors speculated that asthmatic individuals may be deficient for A20 in their lung epithelial cells. However, for some reason, contrary to the authors hypothesis, lung cells from severe asthmatics expressed more A20 molecules compared to lung epithelial cells from mild asthmatics (see figure below). The authors did not discuss this contradiction.

There are several weaknesses in this article. For one, most data are presented as bars rather than scatter plot with individual data points. 2nd, for some unknown reason, the authors decided not to show lung hypersensitivity assay with LPS pre-exposure from mutant mice, as it was done with wild-type mice in Fig. 1D. This assay is clinically the most relevant. This is a Science paper, to remind you.

So, in summary, while I do think that exposure to dirt during early life is relevant for human asthma development, based on results from this paper I am not convinced that expression of A20 in lung epithelial cells play a role here.  

David Usharauli

NSAIDs show synergy with anti-PD1 antibody in cancer immunotherapy

Tumor progression from a single cell to a multicellular state can take years and several mutations that allow cancer cells to (1) expand uncontrollably, and (2) adopt natural defense mechanisms against immune surveillance (commonly referred as tolerance). 

Ordinarily, healthy (or healing) tissues defend themselves against excessive immune probing by secreting or expressing inhibitory molecules. Each healthy tissue will have its own suitable natural defense(s) against excessive immune probing.

There are several known tissue specific inhibitory signals. Many are still unknown. By understanding how healthy tissues communicate tolerogenic signals to immune system we can gain better control over cancer tissue (because cancer tissues are imitating the same tolerogenic state). 

For example, new pepar in prestigious journal Cell showed that blockade of COX1/COX2 enzymes leads to tumor control by adaptive immune system. COX1/COX2 enzymes are the same enzymes the drugs such as aspirin targets to relieve the pain. 

This is very straightforward article. The reason it is in Cell has to due with CRISPR/Cas9 technology the authors has used to develop COX1 or COX1/COX2 double deficient tumor cell lines. The senior author on this paper is Caetano Reis e Sousa, a wellknown name among immunologists. This study was done at the Francis Crick Institute in London which  will be the largest biomedical institution in Europe when completed.

Initially, the authors developed melanoma-prone cell lines (Braf^v600E) deficient for COX enzymes using CRISPR/Cas9 technology.

Next, the authors showed that Braf^v600E melanoma tumor cells deficient for COX1/2 enzymes lack the ability to secrete PGE2 (among other bioactive lipids), a prostaglandin molecule known for its immunomodulating property.

The authors observed that when transplanted COX1/2 DKO melanoma Braf^v600E tumor cells (but not parental cells) were rejected by wild-type, but not by RAG KO hosts, implying the role of adaptive immune system in recognizing tumor cells in absence of PGE2.

Similar results were obtained with melanoma Braf^v600E tumor cells selectively deficient for PGE2 synthase.

Growth of other tumor cell lines (colorectal and breast cancer cells) deficient for COX1/2 enzymes were also controlled by adaptive immune system in wild-type hosts, though less efficiently compared to melanoma cells (the authors do not indicate whether these tumors were ultimately rejected or stayed dormant).

Finally, the authors showed that combination of aspirin (that blocks COX1/2 enzymes) and anti-PD1 antibody could synergize in inhibiting tumor growth (here also it is not clear what term "rejection" means, since tumor growth graph and rejection graph are not identical).

In summary, the data presented in this paper expands our understanding of "natural" defenses of tumor cells against immune probing. Here, tumor growth was controlled by T cells and it could generate memory response to secondary tumor transplantation.

One weakness of this study is the tumor transplantation model itself. The authors did not study already established tumors but rather than they followed the tumor growth or implemented therapy immediately after transplantation. This approach seems to lack a clinical relevance for human cancers where patients are frequently first seen with already established tumors (sometimes quite in advance stages).

David Usharauli