TCF-1 limits intraepithelial lymphocyte antitumor immunity in colorectal carcinoma.

Intraepithelial lymphocytes (IELs), including αß and γδ T cells (T-IELs), constantly survey and play a critical role in maintaining the gastrointestinal epithelium. We show that cytotoxic molecules important for defense against cancer were highly expressed by T-IELs in the small intestine. In contrast, abundance of colonic T-IELs was dependent on the microbiome and displayed higher expression of TCF-1/TCF7 and a reduced effector and cytotoxic profile, including low expression of granzymes. Targeted deletion of TCF-1 in γδ T-IELs induced a distinct effector profile and reduced colon tumor formation in mice. In addition, TCF-1 expression was significantly reduced in γδ T-IELs present in human colorectal cancers (CRCs) compared with normal healthy colon, which strongly correlated with an enhanced γδ T-IEL effector phenotype and improved patient survival. Our work identifies TCF-1 as a colon-specific T-IEL transcriptional regulator that could inform new immunotherapy strategies to treat CRC.

Antioxidant and Anti-Inflammatory Activities of Endemic Plants of the Australian Wet Tropics.

Plants have been a vital source of natural antioxidants since ancient times. Plants growing under various abiotic stress conditions often produce more defensive secondary metabolites such as phenolics, flavonoids, and terpenoids during adaptation to the environment. Many of these secondary metabolites are known to possess antioxidant and anti-inflammatory properties. This study tested seven plants sourced from the mountaintop areas (above 1000 m elevation) of Mount Lewis National Park (falls under the Wet Tropics of Queensland), Australia, for their antioxidant and anti-inflammatory activities. Of the seven studied plants, hydroethanolic extracts of six plants (Leptospermum wooroonooran, Ceratopetalum hylandii, Linospadix apetiolatus, Garcinia brassii, Litsea granitica, and Polyscias willmottii) showed high 2,2-diphenyl-1-picrylhydrazyl (DPPH)-free radical scavenging activity in a dose-dependent (25-1000 µg/mL) manner. At the highest concentration of 1 mg/mL, the DPPH free radical scavenged percentage varied between 75.4% and 92.3%. Only the species Alyxia orophila was inactive in the DPPH free radical scavenging assay. Pseudo-IC50 values of the extracts' ferric reducing antioxidant power (FRAP) based on dose-response curves showed a significant positive correlation with total phenolic content. Five out of the seven plants, namely G. brassii, C. hylandii, L. apetiolatus, L. wooroonooran, and A. orophila, showed inhibitory effects on the secretion of proinflammatory cytokines, tumour necrosis factor (TNF), and interleukins (IL)-23 in a lipopolysaccharide (LPS)-stimulated human peripheral blood mononuclear cells (PBMCs) assay. The results of this study demonstrate the value of tropical mountaintop plants in the biodiscovery of antioxidant and anti-inflammatory lead compounds.

Anti-inflammatory properties of novel galloyl glucosides isolated from the Australian tropical plant Uromyrtus metrosideros.

Two new galloyl glucosides, galloyl-lawsoniaside A (4) and uromyrtoside (6), were isolated from the polar fraction of Uromyrtus metrosideros leaf extract along with another four previously identified phytochemicals (1, 2, 3, and 5). The structures of these six compounds were characterised using low and high-resolution mass spectrometry (L/HRMS) and 1D and 2D Nuclear Magnetic Resonance (NMR) spectroscopy. These compounds were not toxic to human peripheral blood mononuclear cells (PBMCs) at 10 µg/mL over 24 h, yet showed significant in vitro suppression of proinflammatory cytokines involved in the pathogenesis of inflammatory bowel disease (IBD). Specifically, the release of interferon γ (IFN-γ), interleukin (IL)-17A, and IL-8 from phorbol myristate acetate/ionomycin (P/I) and anti-CD3/anti-CD28-activated cells were significantly suppressed by compounds 4 and 5. Interestingly, no effect on tumour necrosis factor (TNF) release was observed. These results show that the newly characterised compound 4 has promising cytokine suppressive properties, which could be further investigated as a candidate for IBD treatment.

MHC Class I on murine hematopoietic APC selects Type A IEL precursors in the thymus.

TCRαß+ CD8α+ CD8ß- intestinal intraepithelial lymphocytes (CD8αα IEL) are gut T cells that maintain barrier surface homeostasis. Most CD8αα IEL are derived from thymic precursors (IELp) through a mechanism referred to as clonal diversion. In this model, self-reactive thymocytes undergo deletion in the presence of CD28 costimulation, but in its absence undergo diversion to the IEL fate. While previous reports showed that IELp were largely ß2m dependent, the APC that drive the development of these cells are poorly defined. We found that both CD80 and CD86 restrain IELp development, and conventional DCs play a prominent role. We sought to define a CD80/86 negative, MHCI positive APC that supports the development to the IEL lineage. Chimera studies showed that MHCI needs to be expressed on hematopoietic APC for selection. As thymic hematopoietic APC are heterogeneous in their expression of MHCI and costimulatory molecules, we identified four thymic APC types that were CD80/86neg/low and MHCI+ . However, selective depletion of ß2m in individual APC suggested functional redundancy. Thus, while hematopoietic APC play a critical role in clonal diversion, no single APC subset is specialized to promote the CD8αα IEL fate.

Autoimmune-Mediated Thymic Atrophy Is Accelerated but Reversible in RelB-Deficient Mice.

Polymorphisms impacting thymic function may decrease peripheral tolerance and hasten autoimmune disease. The NF-κB transcription factor subunit, RelB, is essential for the development and differentiation of medullary thymic epithelial cells (mTECs): RelB-deficient mice have reduced thymic cellularity and markedly fewer mTECs, lacking AIRE. The precise mechanism of this mTEC reduction in the absence of RelB is unclear. To address this, we studied mTECs and dendritic cells (DCs), which critically regulate negative selection, and thymic regulatory T-cells (tTreg) in RelB-/- mice, which have spontaneous multiorgan autoimmune disease. RelB-/- thymi were organized, with medullary structures containing AIRE- mTECs, DCs, and CD4+ thymocytes, but fewer tTreg. Granulocytes infiltrated the RelB-/- thymic cortex, capsule, and medulla, producing inflammatory thymic medullary atrophy, which could be treated by granulocyte depletion or RelB+ DC immunotherapy, with concomitant recovery of mTEC and tTreg numbers. These data indicate that central tolerance defects may be accelerated by autoimmune thymic inflammation where impaired RelB signaling impairs the medullary niche, and may be reversible by therapies enhancing peripheral Treg or suppressing inflammation.

Intravenous Labeling and Analysis of the Content of Thymic Perivascular Spaces.

Following development in the thymus, T cells are thought to exit into the periphery predominantly through perivascular spaces (PVS). This exit route is used by conventional T cells, and likely also applies to unconventional T cell subsets, such as precursors of CD8αα and TCRγδ intraepithelial lymphocytes, regulatory T cells and natural killer T cells. Additional cell types might also be found in the PVS and initiate interactions with exiting T cells. The exact content of the PVS, and the processes within, are not well studied. To distinguish vascular from resident cells within various tissues by flow cytometry, intravenous (i.v.) labeling is becoming a commonly employed method. We recently used anti-CD45.2 antibodies and magnetic enrichment to further evaluate this technique, and compared labeled and unlabeled cells in the thymus and blood. This assay can be used to specifically investigate hematopoietic cell subsets within the PVS of the thymus.

RelB-Deficient Dendritic Cells Promote the Development of Spontaneous Allergic Airway Inflammation.

RelB is a member of the NF-κB family, which is essential for dendritic cell (DC) function and maturation. However, the contribution of RelB to the development of allergic airway inflammation (AAI) is unknown. Here, we identify a pivotal role for RelB in the development of spontaneous AAI that is independent of exogenous allergen exposure. We assessed AAI in two strains of RelB-deficient (RelB-/-) mice: one with a targeted deletion and one expressing a major histocompatibility complex transgene. To determine the importance of RelB in DCs, RelB-sufficient DCs (RelB+/+ or RelB-/-) were adoptively transferred into RelB-/- mice. Both strains had increased pulmonary inflammation compared with their respective wild-type (RelB+/+) and heterozygous (RelB+/-) controls. RelB-/- mice also had increased inflammatory cell influx into the airways, levels of chemokines (CCL2/3/4/5/11/17 and CXCL9/10/13) and T-helper cell type 2-associated cytokines (IL-4/5) in lung tissues, serum IgE, and airway remodeling (mucus-secreting cell numbers, collagen deposition, and epithelial thickening). Transfer of RelB+/- CD11c+ DCs into RelB-/- mice decreased pulmonary inflammation, with reductions in lung chemokines, T-helper cell type 2-associated cytokines (IL-4/5/13/25/33 and thymic stromal lymphopoietin), serum IgE, type 2 innate lymphoid cells, myeloid DCs, γδ T cells, lung Vß13+ T cells, mucus-secreting cells, airway collagen deposition, and epithelial thickening. These data indicate that RelB deficiency may be a key pathway underlying AAI, and that DC-encoded RelB is sufficient to restore control of this inflammation.

High Chlamydia Burden Promotes Tumor Necrosis Factor-Dependent Reactive Arthritis in SKG Mice.

OBJECTIVE: Chlamydia trachomatis is a sexually transmitted obligate intracellular pathogen that causes inflammatory reactive arthritis, spondylitis, psoriasiform dermatitis, and conjunctivitis in some individuals after genital infection. The immunologic basis for this inflammatory response in susceptible hosts is poorly understood. As ZAP-70(W163C) -mutant BALB/c (SKG) mice are susceptible to spondylo-arthritis after systemic exposure to microbial ß-glucan, we undertook the present study to compare responses to infection with Chlamydia muridarum in SKG mice and BALB/c mice. METHODS: After genital or respiratory infection with C muridarum, conjunctivitis and arthritis were assessed clinically, and eye, skin, and joint specimens were analyzed histologically. Chlamydial major outer membrane protein antigen-specific responses were assessed in splenocytes. Treg cells were depleted from FoxP3-DTR BALB/c or SKG mice, and chlamydial DNA was quantified by polymerase chain reaction. RESULTS: Five weeks after vaginal infection with live C muridarum, arthritis, spondylitis, and psoriasiform dermatitis developed in female SKG mice, but not in BALB/c mice. Inflammatory bowel disease did not occur in mice of either strain. The severity of inflammatory disease was correlated with C muridarum inoculum size and vaginal burden postinoculation. Treatment with combination antibiotics starting 1 day postinoculation prevented disease. Chlamydial antigen was present in macrophages and spread from the infection site to lymphoid organs and peripheral tissue. In response to chlamydial antigen, production of interferon-γ and interleukin-17 was impaired in T cells from SKG mice but tumor necrosis factor (TNF) responses were exaggerated, compared to findings in T cells from BALB/c mice. Unlike previous observations in arthritis triggered by ß-glucan, no autoantibodies developed. Accelerated disease triggered by depletion of Treg cells was TNF dependent. CONCLUSION: In the susceptible SKG strain, Chlamydia-induced reactive arthritis develops as a result of deficient intracellular pathogen control, with antigen-specific TNF production upon dissemination of antigen, and TNF-dependent inflammatory disease.