Prevalence of inhibitory LPS within gut microflora could underlie hygiene hypothesis

This week prestigious journal Cell published seminal microbiome study that could explain hygiene hypothesis that suggests that "microbial cleanliness" in developed countries predisposes individuals to immunopathologies such as allergy, multiple sclerosis and other autoimmune diseases. 

This study found that gut microflora in babies from Finland and Estonia, but not from Russian Karelia, are enriched in gut bacteria with inhibitory LPS that failed to properly "educate" newborns innate immune system thus predisposing them to higher incidence of immunopathologies.

The first finding was that microbiota in babies from Finland, but not from Russian Karelia, were enriched in Bacteroides. Now this fact by itself isn't too surprising. 

The second finding was that lipid A component of LPS from these Bacteroides [unlike lipid A component of LPS from E. coli] species were non-stimulatory in TLR4 assay. In fact, molecular analysis showed that Bacteroides harbored tetra- and penta-acylated lipid A structures, as opposed to the hexa-acylated lipid A seen in E. coli. Such structural modification converted Bacteroides lipid A into totally non-stimulatory ligand.

In fact, Bacteroides derived lipid A was inhibitory when combined with stimulatory, E.coli derived lipid A [LPS tolerance assay, where primary exposure to E. coli LPS makes responding cell refractory to secondary exposure].

Finally, third finding was that presence of non-stimulatory lipid A had immunological consequences since it failed to reduce incidence of diabetes in NOD mice [whereas lipid A from E. coli could, as expected].  

In summary, this important study has provided one of the first definite molecular evidence underlying hygiene hypothesis and suggested the path for its prevention.

David Usharauli

     

Making sense of Foxp3 as a prognostic value in tumors

A transcription factor Foxp3 controls functionality of T cell subset called regulatory T cells (T_regs). Seminal studies in mice clearly showed that Foxp3⁺ T cells play a dominant role in suppressing excessive immune response.

But human studies introduced an additional confusion in T_regs field. Unlike, mouse Foxp3, human Foxp3 is up-regulated in human naive CD4 T cells upon activation. In fact, human Foxp3⁺ T cell population is divided into Foxp3^low and Foxp3^high population. Foxp3^low population lack regulatory properties, whereas Foxp3^high population represents a bona fide T_regs cells.

However, such separation of Foxp3 marker within individual tumor clinical samples are not always possible and ordinarily cancer biopsies are grouped into Foxp3^hi or Foxp3^lo for Foxp3 mRNA total expression levels that are above or below the [arbitrary] median. So, it is not surprising that [total] expression level Foxp3 biomarker in tumor tissue does not correlate with overall survival.

In this regard, it is interesting to read a new paper in Nature Medicine showing that inclusion of two additional biomarkers, IL-12 and TGFβ1, could provide that differentiating power to transform Foxp3 expression into a prognostic factor in cancer diagnostics.

For this study the authors analyzed colorectal cancer samples. They found that cancer samples could be grouped into A and B types based on presence of two separate Foxp3⁺ T cell population: Foxp3^low and Foxp3^high population. Type A samples mostly have Foxp3^high population whereas type B samples have both Foxp3^low and Foxp3^high populations.

As expected Foxp3^low T cell population lacked suppressive [cell proliferation inhibition] quality and most likely represent conventional activated T cells.

Gene expression analysis of type A and B cancer tissue showed that type B were enriched with several cytokines such as IL-12, TNF-α and also TGFβ1. In vitro Foxp3 conversion assay showed that when combined with TGFβ1, interleukin-12 [but not TNF-α] could specifically increase naive T cell differentiation into Foxp3^low population (most likely by preventing their differentiation into Foxp3^high population).

Interestingly, when cancer samples were classified based on mRNA expression levels of Foxp3, IL-12 and TGFβ1, there were clear differences in patients survival between groups:

(1) as expected Foxp3 expression by itself had no prognostic value

(2) in samples with IL-12^loTGFβ1^lo expression, mRNA Foxp3^hi was associated with poor prognosis

and (3) unexpectedly, in samples with IL-12^hiTGFβ1^hi expression, mRNA Foxp3^hi was associated with a better prognosis.

Finally, the authors find that cancer samples with IL-12^hiTGFβ1^hi expression harbored more of gut microbe Fusobacterium nucleatum.

In summary, this study indicates following scenario:

(a) in colorectal cancers, detection of mRNA Foxp3 by itself has no prognostic value.
(b) however, cancer biopsies with Foxp3^hiIL-12^loTGFβ1^lo mRNA expression could mean poor prognosis, because such samples contain mostly bona fide T_regs cells.
(c) but, cancer biopsies with Foxp3^lo/hiIL-12^hiTGFβ1^hi mRNA expression could mean good prognosis, because such samples contain mostly Foxp3^low population or it has sufficient inflammatory milieu to override any suppression by bona fide T_regs cells.

David Usharauli

IL-18 contributes to sepsis development during post-natal period

This week PNAS published new study that showed that IL-18 deficiency makes newborn mice resistant to bacterial sepsis. 

Current knowledge about pathophysiology of sepsis is incomplete. In this study, IL-18–null (IL-18−/−) newborn mice showed improved survival when exposed to sepsis-induced bacteria.

Exogenous IL-18 could increase susceptibility of newborn mice to sepsis in absence of adaptive immune system (RAG1−/− mice).

Curiously, TCRδ−/− mice lacking γδT cells [but, unlike RAG1−/− mice, have αβT cells] were resistant to IL-18 effect.

At molecular level, effect of IL-18 on sepsis was dependent of IL-1R signaling 

And also on IL-17 signaling.

In summary, this study expands our knowledge of approaches that can be taken to treat sepsis, for example, Anakinra [an interleukin-1R antagonist] and Cosentyx [anti-interleukin-17A].

David Usharauli

Tumor burden, not cancer per cell antigen density or T cell receptor affinity, determine immunotherapy outcome

The checkpoint inhibitors and adoptive T cell therapies, though quite expensive, are transforming cancer management and outcome. However, even in the best scenario, currently available immunotherapies against few solid tumors work in ~30% of cancer patients. By "work" clinician usually mean treatment when immunotherapy delays patient's death from cancer for  >1 year.

Right now, there is no specific marker that can reliably predict a success or a failure of cancer immunotherapy. So far, best evidence points to a density of cancer-associated mutations as a positive predictor of success in immunotherapy. However, it is too early to generalize this correlation.

For example, following paper published in Journal of Immunology highlights our incomplete understanding of cancer-immune system relationship. Here,  the authors showed that it was overall tumor burden [tumor mass + antigen] rather than cancer [per cell] antigen density or affinity of adoptively transferred T cells that determined outcome of immunotherapy.

For this study the authors used non-Hodgkin mouse lymphoma model, Eµ-myc, engineered to express different amount of OVA antigen. For adoptive T cells, OT-I (high affinity) and OT-3 (low affinity) CD8 T cells were used that differed in their TCR affinity to OVA.

First, the authors injected lymphoma cells and 2 days later transferred high and low affinity T cells. Surprisingly both T cells were able to eliminate lymphoma cells.

Next, the authors repeated the same experiment but this time they transferred T cells 5 days later. This time, both T cells failed to eliminate lymphoma cells.

Moreover, both T cell types exposed to day 5 lymphoma underwent functional "exhaustion" and failed to eliminate antigen-pulsed target cells.

In a separate experiments, the authors also used lymphoma cells expressing different amount of OVA. However, while OT-I could eliminate both types of lymphoma cells when transferred on day 2 post tumor inoculation, the same OT-I were impotent against lymphoma cells when transferred on day 3 post tumor inoculation.

Interestingly, whereas only OT-I CD8 T cells exposed to OVA^high tumor cells down-regulated their TCR receptor, both type of tumors (OVA^high and OVA^low) induced up-regulation of PD1 on OT-I cells.

In summary, this simple study suggests following: neither TCR affinity nor per cell density of mutated antigens determine immunotherapy outcome. Rather it is overall tumor burden [meaning, total level of cancer antigen + total tumor mass] could influence immunotherapy outcome. Another conclusion from this study, though not formally tested, is that co-administration of PD1 checkpoint inhibitors may benefit adoptive T cell therapy.

David Usharauli

Hobit controls tissue residency of memory T cells

Tissue resident memory T cells, T_RM, represent newly recognized T cell population that play physiologically-relevant role in body's defense against reinfection. T_RM cells are local tissue surveyors and act as a first-line defense [alongside of innate immunity] against tissue invading pathogens. Beyond these information nothing much was known about T_RM cells.

Now, a new paper published in Science showed that transcription factor named Hobit is a master regulator of T_RM cell development.   

Using mouse HSV-1 model, the authors found that by day 30 post-infection, Hobit was selectively up-regulated in T_RM cells recovered from peripheral tissues, such as skin, but not in central lymphoid tissues such as spleen.

Using Hobit-KO T cells the authors were able to confirm that Hobit was indeed intimately involved in development of tissue-resident memory T_RM cells (memory stage, day 40+). Deficiency of another transcription factor, Blimp1, had an additive effect on T_RM cells formation when combined with Hobit deficiency. 

Interesting, Hobit, in combination with Blimp1, also controlled tissue residency of innate cells such NK and NKT cells.

In summary, this study revealed that transcription factor Hobit controls tissue residency of memory T cells, T_RM cells. These data should be considered in developing T cell-oriented vaccines, including cancer immunotherapy (one limitation of this study is that for some reason the authors failed to show actual virus protection data with the regard of Hobit or Hobit + Blimp1 DKO T cells. ).

David Usharauli 

New mouse model to mimic immune response to Flu virus in elderly

This week Science published several research papers in immunology. One of these papers is the study from Yale School of Medicine showing that Tlr7^–/– × Mavs^–/– double KO mice expressing human anti-viral molecule Mx1 display susceptibility to influenza A virus in a caspase 1/11-dependent manner.

It is known that elderly individuals show susceptibility to influenza A virus. One reason for this susceptibility is a reduced ability of elderly immune system to produce type I IFNs in response to influenza A virus.

In contrast, aging mice do not show such susceptibility to influenza A virus. Mouse does not express Mx1, a dynamin-like guanosine triphosphatase that blocks primary transcription of influenza in humans. To make mouse model "usable" for  studying human response influenza A virus, the authors created Mx1⁺ mice. Mx1⁺ mice were resistant to experimental influenza A virus infection and this resistance was dependent on Tlr7 and Mavs (both molecules are involved in anti-viral response).

Interestingly, while Mx1⁺ mice double deficient for Tlr7^–/– × Mavs^–/– were susceptible to influenza A virus infection,  Mx1⁺ mice triple deficient for Tlr7^–/– × Mavs^–/–× Casp1/11^–/– were resistant to influenza A virus infection even though both showed similar viral burden (Of note, Tlr7^–/– × Mavs^–/–× Casp1/11^–/– mice were eventually cleared the virus by 30 days after infection).

Moreover, Mx1⁺ mice triple deficient for Tlr7^–/– × Mavs^–/–× Casp1/11^–/– were resistant to influenza A virus infection even though they too showed similar secondary bacterial "bloom" in their airways.

These data suggested that caspase 1/11 signaling reduced "tissue tolerance" [rather than increased anti-viral response] to influenza A virus infection and secondary bacterial "bloom" in Mx1⁺ mice deficient for anti-viral innate signaling via Tlr7^–/– × Mavs^–/–.

In summary, this study showed that intact caspase 1/11 signaling compromises tissue tolerance to acute influenza A virus infection and secondary bacterial "bloom" in host with a weakened anti-viral signaling.

David Usharauli

PAMPs and DAMPs cooperate to drive vigorous adaptive immune response

This week Science published interesting article related to basic question of dendritic cell (DCs) activation and initiation of productive adaptive immune response. It showed that endogenously generated oxidized phospholipids (oxPAPC) cooperate with bacterial-derived LPS in a caspase 1/11 dependent manner to enhance viability and activation status of DCs enabling a better adaptive immune response.  

I would like to note here that title of this paper does not exactly captures the main idea behind this study. In fact, only reading the final portion of the paper one can get the sense and larger meaning of this study. I will try to explain it.   

This paper's main idea is to understand the difference between PAMPs and DAMPs with the regard of their impact on DCs. Every scientists in immunology is familiar with pathogen-associated molecular patterns (PAMPs, such as LPS) and damage-associated molecular patterns (DAMPs, such as ATP). Nonetheless, it is still unclear what role(s), for example, DAMPs play within immune system.

To understand it, the authors focused on one particular type of DAPMs, oxidized phospholipids (oxPAPCs). oxPAPCs are found in inflammatory milieu and can reach concentrations of 10-100 μM in damaged tissues. Initially, the authors showed that unlike LPS, oxPAPCs do not signal via TLR4.

However, similar to other DAMP molecules, oxPAPCs could induce IL-1β release from LPS-primed DCs. As expected, oxPAPC-induced IL-1β release from LPS-primed DCs required inflammasome activation, since such effect were absent in DCs from ASC knockout (KO), caspase-1 KO, caspase-1/caspase-11 double KO or NLRP3 KO mice, each of which are defective for inflammasome functions.

Pyroptosis is an inflammasome-dependent cell death characterized by loss of plasma membrane integrity. Interestingly, unlike LPS or LPS+ATP combination, oxPAPC-induced inflammasomes did not promote pyroptosis. Thus oxPAPC promoted IL-1β release from living DCs.

Finally, the authors showed that mice immunization with antigen in combination with oxPAPC+LPS could improve priming of adaptive T cells in a caspase 11-dependent manner.

In summary, this study showed that DAMP molecule, oxPAPC, promotes DC viability and IL-1β release when combined with PAMP molecule, LPS. Such combination of DAMP and PAMP promoted more productive adaptive immune response.

David Usharauli

Monotherapy with anti-CD47, a "do not eat me" signal", may not be sufficient for cancer immunotherapy

This week PNAS published research article that should be interesting for people following advances in cancer immunotherapy. This new study showed that in mice neither monotherapy with anti-CD47 antibody alone nor its combination with cancer-specific antibody could provide any lasting anti-cancer benefit. However, when combined in addition with anti-PDL1 checkpoint inhibitor, triple combination delivered long-lasting cancer protection.

For this study the authors generated novel high affinity anti-mouse CD47 nanobody that could enhance in vitro cancer cell [melanoma] phagocytosis by macrophages when combined with cancer-specific antibody.

However, in vivo, CD47 nanobody, A4, failed as a (1) monotherapy against melanoma challenge, (b) it couldn't enhance anti-cancer effect when combined with cancer-specific antibody (TA99), (c) it couldn't enhance anti-cancer effect of GM-CSF–producing B16F10 cells vaccine (GVAX) and (d) it slightly improved tumor protection when combined with checkpoint inhibitor PD-L1 antibody.

But, when CD47 nanobody, A4, was combined in triple combination with cancer-specific antibody (TA99) and checkpoint inhibitor PD-L1 antibody, it delivered long-lasting tumor protection in 60% of recipients against primary as well as to secondary tumor challenge indicating tumor-specific memory generation.

In summary, this study revealed that anti-CD47 antibody alone showed minimal activity when used against tumor in hosts with intact immune system (senior author of this study is involved in biotech company focusing on CD47 application). The authors suggested that earlier studies reached different results because they used immunodeficient mouse models [NOD-scid, IL2rgKO (NSG) mice] that lack intact adaptive immune system.

David Usharauli

Type 2 immunity contributes to ECM scaffold-guided muscle tissue regeneration

Few days back Science published a short study suggesting that muscle regeneration post surgical-injury required type 2 immune response orchestrated by adaptive T_H2 cells.

For this study the authors applied three different tissue-derived extracellular matrix (ECM) scaffolds to surgically-injured muscle tissue: (1) particulate collagen, (2) bone–derived tissue ECM scaffold (B-ECM), (3) cardiac muscle–derived tissue ECM scaffold (C-ECM). Gene expression profiling of sorted T cells harvested from muscle tissue 1 week post-injury showed up-regulation of IL-4, a canonical T_H2 cytokine (also IL-17, but the authors did not discuss its significance).

Next, authors found that up-regulation IL-4 in injured tissue was abolished in RAG-KO mice that lack adaptive immune cells, indicating the role of adaptive T_H2 cells in this process.

The role of T_H2 cells in orchestrating type II immune environment was also shown by analysis of CD206⁺ myeloid cells (a mannose receptor and classical M2 marker) from injured tissue in RAG-KO, IL4ra^−/− mice that cannot receive signals from IL-4, or Rictor^−/− CD4⁺ T cells (T-Rictr^−/−, T_H2-deficient).

Finally, the authors showed that functionally-competent T_H2 cells were necessary for wound healing and recovery of muscle functionality in response to cardiac muscle–derived tissue ECM scaffold.

In summary, this short study reveals a complex nature of type 2 immunity. Earlier studies showed that type 2 immunity is [in addition] involved in allergy/asthma, immune response to worms, thermoregulation / lean body metabolism. Now we can to this list tissue regeneration as well.

David Usharauli

Nanoparticle-caged antigens could treat allergen-sensitized host

The challenge with immune system-[driven] disorders is the fact that ordinarily such conditions are detected at the later stages when immune system had already developed self-perpetuating "memory circuit". Even now we know very little how to break such "memory circuit".

New study in PNAS suggested an experimental model that were able to tolerize already-sensitized host against allergen. The authors showed that biodegradable nanoparticles incorporating caged antigen, but not simple conjugates, could deliver both prophylactic and therapeutic treatment to allergen-prone host.

For this study the authors have used widely-accepted experimental sensitization protocol in mice to induce T_H2-driven allergy to ovalbumin  antigen (OVA). Next, they proceeded to test three different combination of  nanoparticles + ovalbumin: (1) OVA conjugated to polystyrene nanoparticles (Ag-PS), (2) OVA conjugated to biodegradable poly(lactide-co-glycolide) nanoparticles (Ag-PLG) and (3) OVA caged within biodegradable poly(lactide-co-glycolide) nanoparticles [PLG(Ag)].

First, the authors found that all three nanoparticle combinations displayed potent prophylactic action against allergen when delivered before allergen sensitization (Ag-PLG is shown here).

However, when applied to already allergen-sensitized host, polystyrene nanoparticles induced severe allergic reaction. In contrast, application of Ag-PLG nanoparticles did not induce allergic response in sensitized hosts and even reduced T_H2 cytokines. However, Ag-PLG nanoparticles could not inhibit lung eosinophilia.

Finally, the authors showed that application of PLG nanoparticles with caged OVA antigen could both prophylactically and therapeutically inhibit T_H2-driven allergic response.

In summary, this study showed that nanoparticles incorporating antigens (Ag caging) avoids detection by allergen-specific immune effector molecules (IgE) and instead drives tolerance to allergen.      

David Usharauli

Worm infection tips the balance in favor of Crohn's disease soothing gut flora

This week Science magazine published yet another study that revealed a complex biological inter-species relationship called defensive symbiosis. Here, the authors showed that worm infection of mammalian host favors gut flora species that soothe intestinal inflammation in a T_H2-dependent manner.

Mice deficient in Nod2 develop small intestinal abnormalities that resemble human Crohn's disease (for example, goblet cell defect that compromises intestinal mucus layer). This new study showed that experimental infection of Nod2-deficient mice with the parasitic worm Trichuris muris (T. muris) could restore small intestinal goblet cell numbers and morphology.

Earlier reports showed that intestinal abnormalities in Nod2-deficient mice dependent on gut flora species, Bacteroides vulgatus. Now, the authors showed that T. muris worm infection could reduce Bacteroides vulgatus burden in Nod2-deficient mice in a manner dependent on STAT6 signaling and IL-13 (type II immunity). Similar results were seen with a second worm infection, Heligmosomoides polygyrus.

Finally, the authors showed that worm infection specifically expanded another gut flora species, Clostridiales, that could directly inhibit pro-inflammatory Bacteroides vulgatus.

In summary, this study showed that in Nod2-deficient mice gut species Clostridiales represent defensive symbionts with an antagonistic interaction with another commensal bacteria, Bacteroidales. Worm infection of Nod2-deficient mice tips the balance in favor anti-inflammatory Clostridiales species. This knowledge could be utilized in treatment of Crohn's disease (for example, therapeutic worm infection or application of its derivative that promote type II immunity). 

David Usharauli

T cell compatible cancer chemotherapy synergizes with cancer adjuvant vaccine

Nowadays it is well acknowledged that optimal cancer treatment would require multi-pronged approach, for example, combining cancer chemotherapy with cancer antigen-specific vaccine to boost tumor-specific T cell response. However, such combination is not always feasible since (a) both tumor and activated T cells are highly proliferative cell types and (b) vast majority of chemotherapy drugs target cellular replication machinery. Basically, it is matter of trial and error to find T cell compatible chemotherapy drugs.

Such combination was recently described in new paper published in Science Translational Medicine. Here, the authors showed that cervical cancer treatment with CarboTaxol (carboplatin + paclitaxel) synergizes with therapeutic T cell vaccine that consisted of HPV16 overlapping synthetic long peptides (HPV16-SLPs) admixed with adjuvant Montanide ISA-51.

Most cervical cancers in humans are induced by human papillomavirus type 16 (HPV16). Initially, the authors showed that CarboTaxol synergized with HPV16-SLP vaccination in HPV16-positive TC-1 tumor-bearing mouse model.

Next, the authors noticed that CarboTaxol [and vaccine too] reduced level of CD11b^highGr1^high myeloid [suppressor] population within tumor tissue.

Similar depletion of myeloid population was observed in blood samples of cervical cancer patients undergoing CarboTaxol therapy.

In fact, CarboTaxol treatment cycles significantly improved T cells proliferation in response to minor [bacterial recall antigen mixture, MRM)] and major HLA antigens [MLR].

Finally, combining CarboTaxol treatment with HPV16-SLP vaccination improved T cell stimulation in response to HPV16 antigens E6/E7.

In summary, this study indicates that CarboTaxol chemotherapy augments, rather than inhibits, tumor-specific T cell priming in response to HPV16-SLP vaccination.

David Usharauli