Label-Free Characterization and Quantification of Mucosal Inflammation in Common Murine Colitis Models With Multiphoton Imaging.

BACKGROUND: Clinical challenges in inflammatory bowel diseases require microscopic in vivo evaluation of inflammation. Here, label-free imaging holds great potential, and recently, our group demonstrated the advantage of using in vivo multiphoton endomicroscopy for longitudinal animal studies. This article extends our previous work by in-depth analysis of label-free tissue features in common colitis models quantified by the multiphoton colitis score (MCS). METHODS: Fresh mucosal tissues were evaluated from acute and chronic dextran sulfate sodium (DSS), TNBS, oxazolone, and transfer colitis. Label-free imaging was performed by using second harmonic generation and natural autofluorescence. Morphological changes in mucosal crypts, collagen fibers, and cellularity in the stroma were analyzed and graded. RESULTS: Our approach discriminated between healthy (mean MCS = 2.5) and inflamed tissue (mean MCS > 5) in all models, and the MCS was validated by hematoxylin and eosin scoring of the same samples (85.2% agreement). Moreover, specific characteristics of each phenotype were identified. While TNBS, oxazolone, and transfer colitis showed high cellularity in stroma, epithelial damage seemed specific for chronic, acute DSS and transfer colitis. Crypt deformations were mostly observed in acute DSS. CONCLUSIONS: Quantification of label-free imaging is promising for in vivo endoscopy. In the future, this could be valuable for monitoring of inflammatory pathways in murine models, which is highly relevant for the development of new inflammatory bowel disease therapeutics.

Small but powerful: will nanoparticles be the future state-of-the-art therapy for IBD?

INTRODUCTION: Inflammatory bowel disease (IBD) is the inflammatory condition of the gastrointestinal tract particularly affecting the colon and the ileum. IBD patients can have a very poor quality of life because of the limited therapeutic efficacy and accompanied adverse effects. AREAS COVERED: The potential ways to employ nanoparticles to deliver drugs to a certain site of inflammation are discussed. The focus was set on the microenvironment in the gut as well as the mucosa, epithelial layer and the microbiota. Moreover, experimental animal colitis models were nanoparticles were used as a potential treatment are presented. Lastly, challenges for the potential clinical use in humans are discussed. EXPERT OPINION: Although there still remain many open questions e.g. regarding the toxicity, the metabolism or the pharmacokinetics of nanoparticles further research on this topic could overcome these challenges. For example, instead of synthetically engineered particles, biodegradable components could be used. Since there have been a lot pf promising results in the recent years, we are sure that in the future nanoparticles will be developed in a way to ensure safe and targeted delivery of drugs to the site of inflammation.

Targeting of the Tec Kinase ITK Drives Resolution of T Cell-Mediated Colitis and Emerges as Potential Therapeutic Option in Ulcerative Colitis.

BACKGROUND & AIMS: The molecular checkpoints driving T cell activation and cytokine responses in ulcerative colitis (UC) are incompletely understood. Here, we studied the Tec kinase ITK in UC. METHODS: We analyzed patients with inflammatory bowel disease (n = 223) and evaluated ITK activity as well as the functional effects of cyclosporine-A (CsA). In addition, 3 independent murine colitis models were used to investigate the functional role of ITK. Finally, the activity of ITK was blocked via pharmacological inhibitors and genetically engineered mice. Readout parameters were mini-endoscopy, histopathology, mucosal T cell apoptosis, and cytokine production. RESULTS: We found an expansion of pITK-expressing mucosal CD4+ T cells in UC rather than Crohn's disease that correlated with disease severity. CsA suppressed activation of ITK in cultured CD4+ T cells and calcineurin-containing microclusters adjacent to the T cell receptor signaling complex. Functionally, the capacity of CsA to suppress activity of experimental colitis was critically dependent on ITK. Genetic inactivation of Itk via gene targeting or induction of allele-sensitive Itk mutants prevented experimental colitis in 3 colitis models, and treatment with pharmacological ITK blockers suppressed established colitis. In addition, ITK controlled apoptosis and activation of mucosal Th2 and Th17 lymphocytes via NFATc2 signaling pathways. CONCLUSIONS: ITK activation was detected in UC and could be down-regulated in cultured T cells by CsA administration. Selective targeting of ITK emerges as an attractive approach for treatment of chronic intestinal inflammation and potentially UC by driving resolution of mucosal inflammation.

The JAK1/3 inhibitor tofacitinib suppresses T cell homing and activation in chronic intestinal inflammation.

BACKGROUND & AIMS: The molecular mechanism of action of the Janus kinase (JAK) inhibitor tofacitinib is poorly understood. METHODS: Here, we analysed the inhibitory effect of tofacitinib on mucosal and blood T cells from patients with ulcerative colitis (UC). Furthermore tofacitinib treatment was analysed in experimental colitis models and wound healing. Additionally, tofacitinib effects were analysed in bioassays. RESULTS: Tofacitinib significantly reduced T cell derived inflammatory cytokine production (Th2, Th9, Th17) in patients with active UC. Additionally, impaired expression of the homing receptors alpha4/beta1 and alpha4/beta7 as well as reduced gut homing capacity of T cells in a humanized mouse model of colitis were observed. Tofacitinib suppressed acute and chronic oxazolone colitis compared to untreated wild-type mice associated with downregulation of cytokines produced by Th2, Th9 and Th17 cells. Functionally, tofacitinib induced apoptosis of intestinal epithelial cells and prevented mucosal wound healing in vivo at higher concentration. Thus, our findings suggest that tofacitinib is quite effective in protecting from colitis by inhibition of a bundle of T cell derived cytokines like IL-5, IL-6, IL-9, IL-13 and IL-17A. CONCLUSION: Application of tofacitinib emerges as an attractive concept for treatment of chronic intestinal inflammation at lower concentrations, whereas higher concentrations require attention due to prolonged wound healing.

Role of the IL-2 inducible tyrosine kinase ITK and its inhibitors in disease pathogenesis.

ITK (IL-2-inducible tyrosine kinase) belongs to the Tec family kinases and is mainly expressed in T cells. It is involved in TCR signalling events driving processes like T cell development as well as Th2, Th9 and Th17 responses thereby controlling the expression of pro-inflammatory cytokines. Studies have shown that ITK is involved in the pathogenesis of autoimmune diseases as well as in carcinogenesis. The loss of ITK or its activity either by mutation or by the use of inhibitors led to a beneficial outcome in experimental models of asthma, inflammatory bowel disease and multiple sclerosis among others. In humans, biallelic mutations in the ITK gene locus result in a monogenetic disorder leading to T cell dysfunction; in consequence, mainly EBV infections can lead to severe immune dysregulation evident by lymphoproliferation, lymphoma and hemophagocytic lymphohistiocytosis. Furthermore, patients who suffer from angioimmunoblastic T cell lymphoma have been found to express significantly more ITK. These findings put ITK in the strong focus as a target for drug development.

NLRP6 Deficiency in CD4 T Cells Decreases T Cell Survival Associated with Increased Cell Death.

The nucleotide-binding oligomerization domain (NOD)-like receptors belong to the family of pattern recognition receptors (PRRs). NOD-like receptors play a role in regulation of innate immune response by recognition of both pathogen-associated molecular patterns that are engulfed during phagocytic process and danger-associated molecular patterns that are mainly byproducts of cell stress mediated response. NOD-like family pyrin domain containing 6 (NLRP6) is one of the 14 pyrin domain-containing receptors. NLRP6 is highly expressed by epithelial and goblet cells to regulate epithelial renewal and mucus production in mice and humans, but its function in T cells is rather unknown. Increased caspase-1 activation and cell death were observed in mouse Nlrp6-deficient T cells following adoptive transfer into Rag2-deficient mice, indicating that Nlrp6 deficiency in CD4+ T cells led to decreased survival.