Dysregulated activation of the innate immune system is a critical driver of chronic inflammation that is associated with at least 30% of all cancers. Innate immunity can also exert tumour-promoting effects (e.g. proliferation) directly on cancer cells in an intrinsic manner. Conversely, innate immunity can influence adaptive immunity-based anti-tumour immune responses via Ag-presenting dendritic cells that activate natural killer and cytotoxic T cells to eradicate tumours. While adaptive anti-tumour immunity has underpinned immunotherapy approaches with immune checkpoint inhibitors and chimeric Ag receptor-T cells, the clinical utility of innate immunity in cancer is underexplored. Innate immune responses are governed by pattern recognition receptors, which comprise several families, including Toll-like, nucleotide-binding oligomerization domain-containing (NOD)-like and absent-in-melanoma 2 (AIM2)-like receptors. Notably, a subset of NOD-like and AIM2-like receptors can form large multiprotein "inflammasome" complexes which control maturation of biologically active IL-1β and IL-18 cytokines. Over the last decade, it has emerged that inflammasomes can coordinate contrasting pro- and anti-tumour responses in cancer and non-cancer (e.g. immune, stromal) cells. Considering the importance of inflammasomes to the net output of innate immune responses, here we provide an overview and discuss recent advancements on the diverse role of inflammasomes in cancer that have underpinned their potential targeting in diverse malignancies.

Helicobacter pylori (H. pylori) infection can trigger chronic gastric inflammation perpetuated by overactivation of the innate immune system, leading to a cascade of precancerous lesions culminating in gastric cancer. However, key regulators of innate immunity that promote H. pylori–induced gastric pathology remain ill‐defined. The innate immune cytosolic DNA sensor absent in melanoma 2 (AIM2) contributes to the pathogenesis of numerous autoimmune and chronic inflammatory diseases, as well as cancers including gastric cancer. We therefore investigated whether AIM2 contributed to the pathogenesis of Helicobacter‐induced gastric disease. Here, we reveal that AIM2 messenger RNA and protein expression levels are elevated in H. pylori–positive versus H. pylori–negative human gastric biopsies. Similarly, chronic Helicobacter felis infection in wild‐type mice augmented Aim2 gene expression levels compared with uninfected controls. Notably, gastric inflammation and hyperplasia were less severe in H. felis–infected Aim2−/− versus wild‐type mice, evidenced by reductions in gastric immune cell infiltrates, mucosal thickness and proinflammatory cytokine and chemokine release. In addition, H. felis–driven proliferation and apoptosis in both gastric epithelial and immune cells were largely attenuated in Aim2−/− stomachs. These observations in Aim2−/− mouse stomachs correlated with decreased levels of inflammasome activity (caspase‐1 cleavage) and the mature inflammasome effector cytokine, interleukin‐1β. Taken together, this work uncovers a pathogenic role for the AIM2 inflammasome in Helicobacter‐induced gastric disease, and furthers our understanding of the host immune response to a common pathogen and the complex and varying roles of AIM2 at different stages of cancerous and precancerous gastric disease.

BACKGROUND & AIMS: Gastric cancer (GC) is strongly linked with chronic gastritis after Helicobacter pylori infection. Toll-like receptors (TLRs) are key innate immune pathogenic sensors that mediate chronic inflammatory and oncogenic responses. Here, we investigated the role of TLR9 in the pathogenesis of GC, including Helicobacter infection. METHODS: TLR9 gene expression was profiled in gastric tis-sues from GC and gastritis patients and from the spontaneous gp130(F/F) GC mouse model and chronic H felis-infected wild-type (WT) mice. Gastric pathology was compared in gp130(F/F) and H felis infection models with or without genetic ablation of Tlr9. The impact of Tlr9 targeting on signaling cascades implicated in inflammation and tumorigenesis (eg, nuclear factor kappa B, extracellular signal-related kinase, and mitogen-activated protein kinase) was assessed in vivo. A direct growth-potentiating effect of TLR9 ligand stimulation on human GC cell lines and gp130(F/F) primary gastric epithelial cells was also evaluated. RESULTS: TLR9 expression was up-regulated in Helicobacter-infected gastric tissues from GC and gastritis patients and gp130(F/F) and H felis-infected WT mice. Tlr9 ablation sup-pressed initiation of tumorigenesis in gp130(F/F):Tlr9(-/-) mice by abrogating gastric inflammation and cellular proliferation. Tlr9(-/-) mice were also protected against H felis-induced gastric inflammation and hyperplasia. The suppressed gastric pathology upon Tlr9 ablation in both mouse models associated with attenuated nuclear factor kappa B and, to a lesser extent, extracellular signal-related kinase, mitogen-activated protein kinase signaling. TLR9 ligand stimulation of human GC cells and gp130(F/F) GECs augmented their proliferation and viability. CONCLUSIONS: Our data reveal that TLR9 promotes the initiating stages of GC and facilitates Helicobacter-induced gastric inflammation and hyperplasia, thus providing in vivo evidence for TLR9 as a candidate therapeutic target in GC.

Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as "two-donor floxing" method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach. Conclusion We propose that the two-donor method lacks efficiency because it relies on two simultaneous recombination events in cis, an outcome that is dwarfed by pervasive accompanying undesired editing events. The methods that use one-donor DNA are fairly efficient as they rely on only one recombination event, and the probability of correct insertion of the donor cassette without unanticipated mutational events is much higher. Therefore, one-donor methods offer higher efficiencies for the routine generation of cKO animal models.

Objective The absent in melanoma 2 (AIM2) cytosolic pattern recognition receptor and DNA sensor promotes the pathogenesis of autoimmune and chronic inflammatory diseases via caspase-1-containing inflammasome complexes. However, the role of AIM2 in cancer is ill-defined. Design The expression of AIM2 and its clinical significance was assessed in human gastric cancer (GC) patient cohorts. Genetic or therapeutic manipulation of AIM2 expression and activity was performed in the genetically engineered gp130 F/F spontaneous GC mouse model, as well as human GC cell line xenografts. The biological role and mechanism of action of AIM2 in gastric tumourigenesis, including its involvement in inflammasome activity and functional interaction with microtubule-associated end-binding protein 1 (EB1), was determined in vitro and in vivo. Results AIM2 expression is upregulated by interleukin-11 cytokine-mediated activation of the oncogenic latent transcription factor STAT3 in the tumour epithelium of GC mouse models and patients with GC. Genetic and therapeutic targeting of AIM2 in gp130 F/F mice suppressed tumourigenesis. Conversely, AIM2 overexpression augmented the tumour load of human GC cell line xenografts. The protumourigenic function of AIM2 was independent of inflammasome activity and inflammation. Rather, in vivo and in vitro AIM2 physically interacted with EB1 to promote epithelial cell migration and tumourigenesis. Furthermore, upregulated expression of AIM2 and EB1 in the tumour epithelium of patients with GC was independently associated with poor patient survival. Conclusion AIM2 can play a driver role in epithelial carcinogenesis by linking cytokine-STAT3 signalling, innate immunity and epithelial cell migration, independent of inflammasome activation.

Enhancers are vitally important during embryonic development to control the spatial and temporal expression of genes. Recently, large scale genome projects have identified a vast number of putative developmental regulatory elements. However, the proportion of these that have been functionally assessed is relatively low. While enhancers have traditionally been studied using reporter assays, this approach does not characterise their contribution to endogenous gene expression. We have studied the murine Nestin (Nes) intron 2 enhancer, which is widely used to direct exogenous gene expression within neural progenitor cells in cultured cells and in vivo. We generated CRISPR deletions of the enhancer region in mice and assessed their impact on Nes expression during embryonic development. Loss of the Nes neural enhancer significantly reduced Nes expression in the developing CNS by as much as 82%. By assessing NES protein localization, we also show that this enhancer region contains repressor element(s) that inhibit Nes expression within the vasculature. Previous reports have stated that Nes is an essential gene, and its loss causes embryonic lethality. We also generated 2 independent Nes null lines and show that both develop without any obvious phenotypic effects. Finally, through crossing of null and enhancer deletion mice we provide evidence of trans-chromosomal interaction of the Nes enhancer and promoter.

Dysregulation of the immune system plays a critical role in driving gastric tumorigenesis, as evidenced by the established link between chronic gastric inflammation triggered by infection with gastric pathogens (i.e., Helicobacter pylori, Epstein–Barr virus) and the onset of gastric cancer. Innate immunity is the first line of host defense against invading microbes and is a source of immunomodulatory factors that can shape the course of gastric tumorigenesis. Innate immune responses are orchestrated by pattern recognition receptors, which comprise several superfamilies, namely, Toll-like receptors, NOD-like receptors, RIG-I-like receptors, C-type lectin receptors, and AIM2-like receptors. Recently, it has emerged that pattern recognition receptors not only coordinate inflammatory responses in immune cells, but also direct pro-tumorigenic cellular processes (e.g., proliferation) in non-immune cells, such as epithelial cells. In this chapter, we will discuss recent advancements on the diverse role of innate immune pattern recognition receptors in gastric cancer that have underpinned their potential targeting as drivers of disease pathogenesis.

Chronic lung diseases including asthma, chronic obstructive pulmonary disease (COPD) and lung fibrosis represent a major burden on healthcare systems with limited effective therapeutic options. Developing effective treatments for these debilitating diseases requires an understanding of how alterations at the molecular level affect lung macroscopic architecture. A common theme among these lung disorders is the presence of an underlying dysregulated immune system which can lead to sustained chronic inflammation. In this respect, several inflammatory cytokines have been implicated in the pathogenesis of lung diseases, thus leading to the notion that cytokines are attractive therapeutic targets for these disorders. In this review, we discuss and highlight the recent breakthroughs that have enhanced our understanding of the role of the interleukin (IL)-6 family of cytokines in lung homeostasis and chronic diseases including asthma, COPD, lung fibrosis and lung cancer.

Mutations in the GAP activity toward RAGs 1 (GATOR1) complex genes (DEPDC5, NPRL2 and NPRL3) have been associated with focal epilepsy and focal cortical dysplasia (FCD). GATOR1 functions as an inhibitor of the mTORC1 signalling pathway, indicating that the downstream effects of mTORC1 deregulation underpin the disease. However, the vast majority of putative disease-causing variants have not been functionally assessed for mTORC1 repression activity. Here, we develop a novel in vitro functional assay that enables rapid assessment of GATOR1-gene variants. Surprisingly, of the 17 variants tested, we show that only six showed significantly impaired mTORC1 inhibition. To further investigate variant function in vivo, we generated a conditional Depdc5 mouse which modelled a ‘second-hit’ mechanism of disease. Generation of Depdc5 null ‘clones’ in the embryonic brain resulted in mTORC1 hyperactivity and modelled epilepsy and FCD symptoms including large dysmorphic neurons, defective migration and lower seizure thresholds. Using this model, we validated DEPDC5 variant F164del to be loss-of-function. We also show that Q542P is not functionally compromised in vivo, consistent with our in vitro findings. Overall, our data show that mTORC1 deregulation is the central pathological mechanism for GATOR1 variants and also indicates that a significant proportion of putative disease variants are pathologically inert, highlighting the importance of GATOR1 variant functional assessment.

Gastric cancer is associated with chronic inflammation (gastritis) triggered by persistent Helicobacter pylori (H. pylori) infection. Elevated tyrosine phosphorylation of the latent transcription factor STAT3 is a feature of gastric cancer, including H. pylori-infected tissues, and aligns with nuclear transcriptional activity. However, the transcriptional role of STAT3 serine phosphorylation, which promotes STAT3-driven mitochondrial activities, is unclear. Here, by coupling serine-phosphorylated (pS)-STAT3-deficient Stat3SA/SA mice with chronic H. felis infection, which mimics human H. pylori infection in mice, we reveal a key role for pS-STAT3 in promoting Helicobacter-induced gastric pathology. Immunohistochemical staining for infiltrating immune cells and expression analyses of inflammatory genes revealed that gastritis was markedly suppressed in infected Stat3SA/SA mice compared with wild-type mice. Stomach weight and gastric mucosal thickness were also reduced in infected Stat3SA/SA mice, which was associated with reduced proliferative potential of infected Stat3SA/SA gastric mucosa. The suppressed H. felis-induced gastric phenotype of Stat3SA/SA mice was phenocopied upon genetic ablation of signaling by the cytokine IL-11, which promotes gastric tumorigenesis via STAT3. pS-STAT3 dependency by Helicobacter coincided with transcriptional activity on STAT3-regulated genes, rather than mitochondrial and metabolic genes. In the gastric mucosa of mice and patients with gastritis, pS-STAT3 was constitutively expressed irrespective of Helicobacter infection. Collectively, these findings suggest an obligate requirement for IL-11 signaling via constitutive pS-STAT3 in Helicobacter-induced gastric carcinogenesis.

Deregulated activation of the latent transcription factor STAT3 has been implicated in the pathogenesis of myeloproliferative and lymphoproliferative hematologic disorders. The uncontrolled activation of STAT3 has traditionally been assigned to its elevated phosphorylation at tyrosine 705 (pY705) and associated nuclear transcriptional activity. By contrast, a transcriptional role for serine 727 phosphorylation (pS727) of STAT3 has recently emerged, suggesting that pS727 may account for the pathological activity of STAT3 in certain disease settings. Here, by coupling pS727-STAT3-deficient Stat3SA/SA mice with a STAT3-driven mouse model (gp130F/F) for myeloproliferative and lymphoproliferative pathologies, we reveal a key role for pS727-STAT3 in promoting multiple hematologic pathologies. The genetic blockade of pS727-STAT3 in gp130F/F:Stat3SA/SA mice ameliorated the neutrophilia, thrombocytosis, splenomegaly and lymphadenopathy that are features of gp130F/F mice. The protection against thrombocytosis in gp130F/F:Stat3SA/SA mice coincided with normalized megakaryopoiesis in both bone marrow and spleen compartments. Interestingly, pS727-STAT3-mediated abnormal lymphopoiesis in gp130F/F mice was more pronounced in lymph nodes compared to thymus, and was characterized by elevated numbers of B cells at the expense of T cells. Furthermore, pS727-STAT3 dependency for these hematologic pathologies coincided with transcriptional activity on STAT3-regulated genes, rather than its effect on mitochondrial and metabolic genes. Collectively, these findings suggest that pS727 plays a critical pathological role in modulating the transcriptional activity of STAT3 in hematologic disorders.

Table S3. The results of the two-donor floxing approach via electroporation for 7 unique loci. (XLSX 11 kb)

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CRISPR-Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA-2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA-2ssODN approach, we performed a generalized linear regression model. We show that factors such as the concentration of the sgRNA, Cas9 protein or the distance between the placement of LoxP insertions were not predictive for the success of this technique. The major predictor affecting the method’s success was the probability of simultaneously inserting intact proximal and distal LoxP sequences, without the loss of the DNA segment between the two sgRNA cleavage sites. Our analysis of a large data set indicates that the 2sgRNA–2ssODN method generates a large number of undesired alleles (>99%), and a very small number of desired alleles (<1%) requiring, on average 1,192 zygotes.

DEPDC5 mutations have recently been shown to cause epilepsy in humans. Evidence from in vitro studies has implicated DEPDC5 as a negative regulator of mTORC1 during amino acid insufficiency as part of the GATOR1 complex. To investigate the role of DEPDC5 in vivo we generated a null mouse model using targeted CRISPR mutagenesis. Depdc5 homozygotes display severe phenotypic defects between 12.5-15.5 dpc, including hypotrophy, anaemia, oedema, and cranial dysmorphology as well as blood and lymphatic vascular defects. mTORC1 hyperactivity was observed in the brain of knockout embryos and in fibroblasts and neurospheres isolated from knockout embryos and cultured in nutrient deprived conditions. Heterozygous mice appeared to be normal and we found no evidence of increased susceptibility to seizures or tumorigenesis. Together, these data support mTORC1 hyperactivation as the likely pathogenic mechanism that underpins DEPDC5 loss of function in humans and highlights the potential utility of mTORC1 inhibitors in the treatment of DEPDC5-associated epilepsy.