Long-term exposure to house dust mites accelerates lung cancer development in mice.

BACKGROUND: Individuals with certain chronic inflammatory lung diseases have a higher risk of developing lung cancer (LC). However, the underlying mechanisms remain largely unknown. Here, we hypothesized that chronic exposure to house dust mites (HDM), a common indoor aeroallergen associated with the development of asthma, accelerates LC development through the induction of chronic lung inflammation (CLI).  METHODS: The effects of HDM and heat-inactivated HDM (HI-HDM) extracts were evaluated in two preclinical mouse models of LC (a chemically-induced model using the carcinogen urethane and a genetically-driven model with oncogenic KrasG12D activation in lung epithelial cells) and on murine macrophages in vitro. Pharmacological blockade or genetic deletion of the Nod-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome, caspase-1, interleukin-1ß (IL-1ß), and C-C motif chemokine ligand 2 (CCL2) or treatment with an inhaled corticosteroid (ICS) was used to uncover the pro-tumorigenic effect of HDM.  RESULTS: Chronic intranasal (i.n) instillation of HDM accelerated LC development in the two mouse models. Mechanistically, HDM caused a particular subtype of CLI, in which the NLRP3/IL-1ß signaling pathway is chronically activated in macrophages, and made the lung microenvironment conducive to tumor development. The tumor-promoting effect of HDM was significantly decreased by heat treatment of the HDM extract and was inhibited by NLRP3, IL-1ß, and CCL2 neutralization, or ICS treatment. CONCLUSIONS: Collectively, these data indicate that long-term exposure to HDM can accelerate lung tumorigenesis in susceptible hosts (e.g., mice and potentially humans exposed to lung carcinogens or genetically predisposed to develop LC).

The TRPA1 ion channel is expressed in CD4+ T cells and restrains T-cell-mediated colitis through inhibition of TRPV1.

OBJECTIVE: Transient receptor potential ankyrin-1 (TRPA1) and transient receptor potential vanilloid-1 (TRPV1) are calcium (Ca2+)-permeable ion channels mostly known as pain receptors in sensory neurons. However, growing evidence suggests their crucial involvement in the pathogenesis of IBD. We explored the possible contribution of TRPA1 and TRPV1 to T-cell-mediated colitis. DESIGN: We evaluated the role of Trpa1 gene deletion in two models of experimental colitis (ie, interleukin-10 knockout and T-cell-adoptive transfer models). We performed electrophysiological and Ca2+ imaging studies to analyse TRPA1 and TRPV1 functions in CD4+ T cells. We used genetic and pharmacological approaches to evaluate TRPV1 contribution to the phenotype of Trpa1-/- CD4+ T cells. We also analysed TRPA1 and TRPV1 gene expression and TRPA1+TRPV1+ T cell infiltration in colonic biopsies from patients with IBD. RESULTS: We identified a protective role for TRPA1 in T-cell-mediated colitis. We demonstrated the functional expression of TRPA1 on the plasma membrane of CD4+ T cells and identified that Trpa1-/- CD4+ T cells have increased T-cell receptor-induced Ca2+ influx, activation profile and differentiation into Th1-effector cells. This phenotype was abrogated upon genetic deletion or pharmacological inhibition of the TRPV1 channel in mouse and human CD4+ T cells. Finally, we found differential regulation of TRPA1 and TRPV1 gene expression as well as increased infiltration of TRPA1+TRPV1+ T cells in the colon of patients with IBD. CONCLUSIONS: Our study indicates that TRPA1 inhibits TRPV1 channel activity in CD4+ T cells, and consequently restrains CD4+ T-cell activation and colitogenic responses. These findings may therefore have therapeutic implications for human IBD.

The ion channel TRPV1 regulates the activation and proinflammatory properties of CD4⁺ T cells.

TRPV1 is a Ca(2+)-permeable channel studied mostly as a pain receptor in sensory neurons. However, its role in other cell types is poorly understood. Here we found that TRPV1 was functionally expressed in CD4(+) T cells, where it acted as a non-store-operated Ca(2+) channel and contributed to T cell antigen receptor (TCR)-induced Ca(2+) influx, TCR signaling and T cell activation. In models of T cell-mediated colitis, TRPV1 promoted colitogenic T cell responses and intestinal inflammation. Furthermore, genetic and pharmacological inhibition of TRPV1 in human CD4(+) T cells recapitulated the phenotype of mouse Trpv1(-/-) CD4(+) T cells. Our findings suggest that inhibition of TRPV1 could represent a new therapeutic strategy for restraining proinflammatory T cell responses.

Signal transducer and activator of transcription 3 (STAT3) protein suppresses adenoma-to-carcinoma transition in Apcmin/+ mice via regulation of Snail-1 (SNAI) protein stability.

STAT3 was recently reported to suppress tumor invasion in Apc(min)(/+) mice. We investigated the mechanisms by which STAT3 inhibits intestinal epithelial tumors using Apc(min)(/+)/Stat3(IEC-KO) mice (intestinal epithelial cell (IEC)-specific deletion of STAT3 in the Apc(min)(/+) background) to determine the role of STAT3 in carcinogenesis in vivo as well as colorectal cancer cell lines in vitro. To inhibit invasion of IEC tumors, STAT3 functions as a molecular adaptor rather than a transcription factor. Accordingly, the tumors in Apc(min)(/+)/Stat3(IEC-KO) mice undergo adenoma-to-carcinoma transition and acquire an invasive phenotype. Similarly, STAT3 knockdown in a colorectal cell line enhances IEC invasion. We demonstrate that STAT3 down-regulates SNAI (Snail-1) expression levels and hence suppresses epithelial-mesenchymal transition of colorectal cancer cells. Mechanistically, STAT3 facilitates glycogen synthase kinase (GSK) 3ß-mediated degradation of SNAI by regulating phosphorylation of GSK3ß. Our data identified a new role for STAT3 in the adenoma-to-carcinoma sequence of intestinal tumors.

ERK activation drives intestinal tumorigenesis in Apc(min/+) mice.

Toll-like receptor (TLR) signaling is essential for intestinal tumorigenesis in Apc(min/+) mice, but the mechanisms by which Apc enhances tumor growth are unknown. Here we show that microflora-MyD88-ERK signaling in intestinal epithelial cells (IECs) promotes tumorigenesis by increasing the stability of the c-Myc oncoprotein. Activation of ERK (extracellular signal-related kinase) phosphorylates c-Myc, preventing its ubiquitination and subsequent proteasomal degradation. Accordingly, Apc(min/+)/Myd88(-/-) mice have lower phospho-ERK (p-ERK) levels and fewer and smaller IEC tumors than Apc(min/+) mice. MyD88 (myeloid differentiation primary response gene 88)-independent activation of ERK by epidermal growth factor (EGF) increased p-ERK and c-Myc and restored the multiple intestinal neoplasia (Min) phenotype in Apc(min/+)/Myd88(-/-) mice. Administration of an ERK inhibitor suppressed intestinal tumorigenesis in EGF-treated Apc(min/+)/Myd88(-/-) and Apc(min/+) mice and increased their survival. Our data reveal a new facet of oncogene-environment interaction, in which microflora-induced TLR activation regulates oncogene expression and related IEC tumor growth in a susceptible host.

Use of recombinant Entamoeba histolytica cysteine proteinase 1 to identify a potent inhibitor of amebic invasion in a human colonic model.

Cysteine proteinases are key virulence factors of the protozoan parasite Entamoeba histolytica. We have shown that cysteine proteinases play a central role in tissue invasion and disruption of host defenses by digesting components of the extracellular matrix, immunoglobulins, complement, and cytokines. Analysis of the E. histolytica genome project has revealed more than 40 genes encoding cysteine proteinases. We have focused on E. histolytica cysteine proteinase 1 (EhCP1) because it is one of two cysteine proteinases unique to invasive E. histolytica and is highly expressed and released. Recombinant EhCP1 was expressed in Escherichia coli and refolded to an active enzyme with a pH optimum of 6.0. We used positional-scanning synthetic tetrapeptide combinatorial libraries to map the specificity of the P1 to P4 subsites of the active site cleft. Arginine was strongly preferred at P2, an unusual specificity among clan CA proteinases. A new vinyl sulfone inhibitor, WRR483, was synthesized based on this specificity to target EhCP1. Recombinant EhCP1 cleaved key components of the host immune system, C3, immunoglobulin G, and pro-interleukin-18, in a time- and dose-dependent manner. EhCP1 localized to large cytoplasmic vesicles, distinct from the sites of other proteinases. To gain insight into the role of secreted cysteine proteinases in amebic invasion, we tested the effect of the vinyl sulfone cysteine proteinase inhibitors K11777 and WRR483 on invasion of human colonic xenografts. The resultant dramatic inhibition of invasion by both inhibitors in this human colonic model of amebiasis strongly suggests a significant role of secreted amebic proteinases, such as EhCP1, in the pathogenesis of amebiasis.

An unusual surface peroxiredoxin protects invasive Entamoeba histolytica from oxidant attack.

Peroxiredoxins are an important class of antioxidant enzymes found from Archaea to humans, which reduce and thereby detoxify peroxides and peroxynitrites. The major thiol-containing surface antigen of the invasive ameba, Entamoeba histolytica, is a peroxiredoxin and is likely to be important during the transition from the anaerobic environment of the large intestine to human tissues. The closely related species, Entamoeba dispar, is incapable of invasion and more sensitive to hydrogen peroxide, yet also has a peroxiredoxin. We cloned and expressed the two active recombinant enzymes and found that their activity was similar by a fluorometric stopped-flow assay, giving a Km of <10 microM for hydrogen peroxide. Three monoclonal antibodies produced to recombinant E. histolytica peroxiredoxin cross-reacted with Entamoeba dispar.E. histolytica contains as much as 50 times more peroxiredoxin than E. dispar as demonstrated by a sensitive capture ELISA. In addition, the peroxiredoxin is present largely on the outer surface of the cell, in contrast to E. dispar. This unusual peroxiredoxin localizes to the site of parasite-host cell contact where it can effectively counteract oxidants generated by host cells, thus facilitating invasion.

Cysteine proteinases from distinct cellular compartments are recruited to phagocytic vesicles by Entamoeba histolytica.

Cysteine proteinases, which are encoded by at least seven genes, play a critical role in the pathogenesis of invasive amebiasis caused by Entamoeba histolytica. The study of these enzymes has been hampered by the inability to obtain significant quantities of the individual native proteinases. We have now expressed functionally active recombinant ACP1 (EhCP3) and ACP2 (EhCP2) proteinases in baculoviral expression vectors. The purified recombinant ACP1 and ACP2 proteinases exhibited similar activities for fluorogenic peptide substrates, especially in their preference for an arginine residue at the P2 position. Although ACP1 and ACP2 are structurally cathepsin L, homology modeling revealed that the aspartic acid in the S2 pocket would result in a substrate specificity for positively charged amino acids, like cathepsin B. The hydrolysis of peptide substrates was strongly inhibited by small peptidyl inhibitors specifically designed for parasitic cysteine proteinases. Confocal and immunoelectron microscopy localization of the proteinases with monoclonal and monospecific antibodies raised to the recombinant enzymes and peptides demonstrated that ACP2 was membrane-associated while ACP1 was cytoplasmic. Following phagocytosis of erythrocytes, ACP1, as well as the membrane-associated cysteine proteinase, ACP2, were incorporated into phagocytic vesicles. These studies suggest that E. histolytica has a redundancy of cysteine proteinases for intracellular digestion and that they may be recruited from different cellular compartments to the site of digestion of phagocytosed cells. The production of active proteinases in baculovirus and large scale recombinant enzymes in bacteria should further our understanding of the role of different cysteine proteinase gene products in virulence.