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  

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

Apoptosis sensing receptors protect brain from virus invasion

The TAM receptors, Tyro3, Axl, and Mertk, and their ligands, Gas6 and Protein S, are essential for the efficient phagocytosis of apoptotic cells. In the immune system, they act as inhibitors of the innate inflammatory response to pathogens. It was hypothesized that deletion of TAM receptors in dendritic cells could improve anti-viral immunity.  

Unexpectedly, however, new study from Nature Medicine showed that deletion of TAM receptors increased mice susceptibility to neurotropic viruses by its effect on blood-brain barrier (BBB).

Due to complex nature of TAM receptors biology, I am reviewing this paper as an example of "surprising finding" rather than an example of definite proof of concept. I will just quickly go through more or less straightforward findings of this paper. 

First, the authors found that TAM receptor deletion made mice highly susceptible to West Nile virus (WNV) subQ infection (no difference were seen for viral pfu/g with direct intra-brain infection). 

Next, TAM deficient mice were also susceptible to another neurotropic virus, La Crosse virus (LACV).

Finally, the authors showed that TAM receptor deficiency increased BBB permeability.

In summary, the results from this study were unexpected. The authors pointed out that development of TAM inhibitors (e.g. clinicaltrials.gov ID:NCT00605618) could be affected by these findings.

David Usharauli

Neither apoptosis nor necrosis but necroptosis allows cross-priming of CD8 T cells

Some pathogens, especially viruses, infect only epithelial cells but not dendritic cells (DCs). This creates dilemma for immune system since activation of naive CD8 T cells require dendritic cells. So how can DCs present cytoplasmic [viral] antigens to CD8 T cells without having one?

This process is called cross-presentation. Since discovery of programmed cell death called apoptosis or silent cell death, it was proposed that apoptosis-derived antigens were cross-presented for tolerance while necrosis-derived antigens [from virus infected cells] were cross-presented for priming. However, data were not consistent and frequently contradictory results were produced. 

Now new paper in journal Science provided support for the alternative model for cross-presentation that could reconcile and explain earlier observations. It found that it is 3rd pathway, called programmed necrosis or necroptosis, rather than apoptosis or necrosis, that provides cargo proteins for cross-presentation by DCs.

To study effect of apoptosis or necroptosis, the authors transduced NIH-3T3 cell line with fusion constructs containing caspase 8 (involved in apoptosis), or RIPK3 (involved in ripoptosome-mediated necroptosis) or RIPK3ΔC (inducing cell death without ripoptosome). When exposed to dimerization reagent, these transduced cells underwent cell death according to the predicted pathway.

Next, the authors showed that dimerization of RIPK3 (referred here as acR3) or RIPK3ΔC (referred here as ac3ΔC) constructs, but not caspase 8, could induce release of damage-associate molecular patterns (DAMPs) such as HMGB1 or ATP.

More importantly, however, when these transduced cell lines [also expressing OVA antigen] were injected into mice to induce CD8 T cell response, the authors found that only wild-type RIPK3 construct-induced cell death promoted CD8 T cell expansion and effector differentiation.

In addition, the authors found that neither secondary necrosis nor mechanical [freeze-thaw] necrosis could prime CD8 T cells.

Next, the authors observed that cells undergoing RIPK3-ripoptsome mediated necroptosis selectively release IL-6 and show rapid RIPK1-mediated IκB degradation.

Finally, using CRISPR/cas9 modified CT26 tumor cell line, the authors showed that RIPK3-mediated ripoptosome assembly involving RIPK1-NF-κB pathway was crucial for immunogenic necroptosis in tumor challenge model.

In summary, this study further refined our understanding of immunogenic cell death and further defined molecular components essential to achieve it. Of note, the role of necroptosis in immunogenic cell death may finally harmonize prior data regarding necrosis or apoptosis in cross-presentation and improve our therapeutic tool box.

David Usharauli

          

Caspase activity determines release of endogenous danger signal during apoptosis

Apoptosis is a fundamental biological process necessary for maintenance of normal cellular turnover. Initially, apoptosis, as a "voluntary" cell death was contrasted to necrosis, "involuntary" cell death. More recently, however, additional forms of cell death were described sharing characteristics of both of these processes (necroptosis, pyroptosis).

From an immunological point of view, cell death could be classified as a silent, non-immunogenic or a noisy, immunogenic. Interestingly, many anti-cancer therapy drugs were shown to induce apoptotic cell death that were immunogenic. Very recently, DNA detecting pathway involving IFN-beta (as an endogenous danger factor), cGAS, STING and IRF3 were shown to contribute to tumor cell detection by immune system following cancer cell apoptosis.

The two new studies in journal Cell provided additional results that may explain some long standing observations about apoptosis. I am going to review both of them separately.

First study was led by Prof Benjamin Kile at the Walter and Eliza Hall Institute of Medical Research, Australia, and Michael White (postdoctoral fellow at his lab) as a first author.

This group studied a role of apoptosis in physiology of haematopoiesis (not a typical immunology research) and it seems they accidentally came to the thought-provoking finding.

While working on apoptotic pathways involving BAX/BAK and caspase 9, the authors made an observation that in bone marrow chimera (BM) mice, caspase 9-KO donor haematopoietic stem cells yielded more lineage-negative stem cells compared to BAX/BAK DKO or wt donor cells.

Analysis indicated that IFN-beta was specifically up-regulated in caspase 9-KO BM chimera. The authors reasoned that there was a connection.  

Indeed, BM chimera mice transplanted with caspase 9-KO BM cells on IFN-receptor alpha 1 deficient background abolished this effect.

To better understand the connection between caspase 9 and IFN-beta, the authors induced mouse splenocytes apoptosis in presence of caspase inhibitor. Unexpectedly, in presence of caspase inhibitor, cells secreted IFN-beta when exposed to pro-apoptotic stimulus.

Similar observation was made with human peripheral blood mononuclear cells (PBMC).

To analysis of sera from mice deficient in different molecules in apoptotic pathway showed that mice  deficient of pro-apoptotic caspases (caspase 9, caspase 3/7) had high levels of serum IFN-beta.

The authors reasoned that in absence of active caspase 9 (or during its inhibition), an initial pro-apoptotic stimulus induce release of mitochondrial DNA (mtDNA) which is recognized by cytosolic DNA sensors, like STING, leading to IFN-beta production. Indeed, pro-apoptotic stimulus could not induce IFN-beta secretion in cells lacking mtDNA even in presence of caspase inhibitors.

In addition, experiments with STING-KO cells, or with CRISPR-Cas9 targeted cGAS -KO and IRF3-KO clones confirmed that no IFN-beta was produced in absence of STING pathway.

Finally, immunoprecipitation of cGAS followed by PCR amplification of co-precipitated DNA confirmed that mtDNA, but not genomic DNA, was enriched with cGAS in treatment group.

In summary, these results indicated that pro-apoptotic stimuli induce mtDNA release, probably as a bystander product of mitochondria membrane permeabilization, which can interact with STING pathway. However, normally, caspase 9 (and caspase 3 and 7) prevent mtDNA from being detected by STING pathway. It is not clear yet how is this accomplished. 

This finding may provide new understanding of immunogenic versus non-immunogenic cell death and reconcile several prior observations relevant for cancer immunotherapy. 

I would like to see the following experiments: use of physiological apoptotic stimuli, like FasL, TNF-alpha, TRAIL, granzyme B, rather than chemical molecules; Also in Fig 5I, pan-caspase inhibitor increased IFN-beta production even in caspase 9KO or caspase 3/7 DKO cells, implying that other caspases are maybe involved too.

David Usharauli