Anti-ß7 integrin treatment impedes the recruitment on non-classical monocytes to the gut and delays macrophage-mediated intestinal wound healing.

BACKGROUND: Closing mucosal defects to reach mucosal healing is an important goal of therapy in inflammatory bowel disease (IBD). Among other cells, monocyte-derived macrophages are centrally involved in such intestinal wound healing. We had previously demonstrated that the anti-α4ß7 integrin antibody vedolizumab blocks the recruitment of non-classical monocytes as biased progenitors of wound healing macrophages to the gut and delays wound healing. However, although important for the interpretation of disappointing results in recent phase III trials in IBD, the effects of the anti-ß7 antibody etrolizumab on wound healing are unclear so far. METHODS: We analyzed the expression of etrolizumab targets on human and mouse monocyte subsets by flow cytometry and assessed their function in adhesion and homing assays. We explored wound-associated monocyte recruitment dynamics with multi-photon microscopy and compared the effects of etrolizumab and vedolizumab surrogate (-s) antibodies on experimental wound healing and wound-associated macrophage abundance. Finally, we investigated wound healing macrophage signatures in the large intestinal transcriptome of patients with Crohn's disease treated with etrolizumab. RESULTS: Human and mouse non-classical monocytes expressed more αEß7 integrin than classical monocytes and were a target of etrolizumab-s, which blocked non-classical monocyte adhesion to MAdCAM-1 and E-Cadherin as well as gut homing in vivo. Intestinal wound healing was delayed on treatment with etrolizumab-s along with a reduction of peri-lesional wound healing macrophages. Wound healing macrophage signatures in the colon of patients with Crohn's disease were substantially down-regulated on treatment with etrolizumab, but not with placebo. CONCLUSIONS: Combined blockade of αEß7 and α4ß7 with etrolizumab seems to exceed the effect of anti-α4ß7 treatment on intestinal wound healing, which might help to inform further investigations to understand the recent observations in the etrolizumab phase III trial program.

Nr4a1-dependent non-classical monocytes are important for macrophage-mediated wound healing in the large intestine.

Introduction: Macrophages play an important role in intestinal wound healing. However, the trajectories from circulating monocytes to gut macrophages are incompletely understood. Methods: Taking advantage of mice depleted for non-classical monocytes due to deficiency for the transcription factor Nr4a1, we addressed the relevance of non-classical monocytes for large intestinal wound healing using flow cytometry, in vivo wound healing assays and immunofluorescence. Results: We show that wound healing in Nr4a1-deficient mice is substantially delayed and associated with reduced peri-lesional presence of macrophages with a wound healing phenotype. Discussion: Our data suggest that non-classical monocytes are biased towards wound healing macrophages. These insights might help to understand, how targeting monocyte recruitment to the intestine can be used to modulate intestinal macrophage functions.

Intestinal Mucosal Wound Healing and Barrier Integrity in IBD-Crosstalk and Trafficking of Cellular Players.

The intestinal epithelial barrier is carrying out two major functions: restricting the entry of potentially harmful substances while on the other hand allowing the selective passage of nutrients. Thus, an intact epithelial barrier is vital to preserve the integrity of the host and to prevent development of disease. Vice versa, an impaired intestinal epithelial barrier function is a hallmark in the development and perpetuation of inflammatory bowel disease (IBD). Besides a multitude of genetic, molecular and cellular alterations predisposing for or driving barrier dysintegrity in IBD, the appearance of intestinal mucosal wounds is a characteristic event of intestinal inflammation apparently inducing breakdown of the intestinal epithelial barrier. Upon injury, the intestinal mucosa undergoes a wound healing process counteracting this breakdown, which is controlled by complex mechanisms such as epithelial restitution, proliferation and differentiation, but also immune cells like macrophages, granulocytes and lymphocytes. Consequently, the repair of mucosal wounds is dependent on a series of events including coordinated trafficking of immune cells to dedicated sites and complex interactions among the cellular players and other mediators involved. Therefore, a better understanding of the crosstalk between epithelial and immune cells as well as cell trafficking during intestinal wound repair is necessary for the development of improved future therapies. In this review, we summarize current concepts on intestinal mucosal wound healing introducing the main cellular mediators and their interplay as well as their trafficking characteristics, before finally discussing the clinical relevance and translational approaches to therapeutically target this process in a clinical setting.

Differentiation of European and Chinese Truffle (Tuber sp.) Species by Means of Sterol Fingerprints.

The increasing demand of valuable truffles (Tuber sp.) has prompted new areas of naturally growing truffles entering the market. Hence, the identification of valueless Tuber species is an important task to prevent food fraud. Here, we show that sterol patterns are suited to differentiate five Tuber species (Tuber magnatum, Tuber melanosporum, Tuber aestivum, Tuber albidum, and Tuber indicum varieties) from each other. Next to the known main sterols of Tuber, ergosterol and brassicasterol, occurrence of minor sterols in differing shares resulted in characteristic fingerprints in the five Tuber species, irrespective of the country of origin. A total of 27 sterols were evaluated, and we proposed assignment criteria of main sterol relations as well as eight distinct biomarkers within the minor compounds for the differentiation of European and Chinese truffles.

Transport and fate of aflibercept in VEGF-A165-challenged retinal endothelial cells.

Retinal vessels are at least in part involved in clearing of Fc terminus-containing proteins from the vitreous. In vitro, the Fc fusion protein aflibercept is transported through a monolayer of unchallenged immortalized bovine retinal endothelial cells (iBREC), mediated by the neonatal Fc receptor (FcRn), but part of the Fc fusion protein is also degraded. Aflibercept's target VEGF-A not only enhances the permeability of REC by destabilization of tight junctions (TJs) thereby allowing for paracellular flow, it may also lower the intracellular stability of the Fc fusion protein by changing its binding properties to the FcRn. Therefore, we investigated the transport and fate of aflibercept in VEGF-A165-challenged iBREC. All cell culture media were supplemented with 5% fetal bovine serum (FBS) as its absence results in accumulation of aflibercept in iBREC due to deregulated expression of transport proteins. Early after exposure of a confluent iBREC monolayer cultivated on gold electrodes to 5% FBS, the cell index (CI) - assessed as a measure of barrier function, cell viability and cell adhesion - transiently declined but recovered again within a few hours to high values. These values remained stable for several days associated with a strong expression of the TJ-protein claudin-1, indicative of a functional barrier formed by the iBREC monolayer. Transient changes of the plasma membrane localizations of claudin-5 and vascular endothelial cadherin - both important for regulation of paracellular flow - accompanied the transient reduction of the CI not prevented by VEGF-binding proteins. Treatment of iBREC with 50 ng/ml VEGF-A165 for one day resulted in a strong and persistent decline of the CI associated with a low expression level of the TJ-protein claudin-1; reversion to normal values was complete one day after aflibercept's addition at a final concentration of 250 µg/ml. Expressions of other proteins involved in regulation of paracellular flow or transcellular transport were not significantly changed. More aflibercept passed through the monolayer of iBREC cultivated on permeable membrane inserts pretreated with VEGF-A for one day, but this was not affected by a FcRn-inhibiting antibody. Subcellular localization of aflibercept was hardly changed in VEGF-A-exposed iBREC 3 h after its addition to the cells; inhibition of (non)-lysosomal or proteasomal proteases then only weakly affected the amount of internalized aflibercept. iBREC also internalized VEGF-A which was barely detectable as early as 2 h after addition of aflibercept. In contrast, blocking the tyrosine kinase activity of VEGF receptor(s) did not prevent VEGF-A's uptake. Inhibition of cellular proteases strongly increased the amount of internalized VEGF-A in the absence and presence of the Fc fusion protein. We therefore conclude that a FcRn-mediated transport plays a minor role in aflibercept's passage through a leaky barrier of REC. Even early after addition of aflibercept to VEGF-A-exposed iBREC, the levels of free intracellular VEGF-A are low, as aflibercept likely prevents binding of VEGF-A to its receptor. Interestingly, the growth factor's detrimental effects still persist for nearly one day.

Total Recall: Intestinal TRM Cells in Health and Disease.

Tissue-resident memory T cells (TRM cells) have crucial functions in host defense in mucosal tissues. They provide local adaptive immune surveillance and allow the fast initiation of targeted adaptive immune responses in case of antigen re-exposure. Recently, an aberrant activation in the case of immunologically mediated diseases has been increasingly acknowledged. As the organ with the largest interface to the environment, the gastrointestinal tract faces billions of antigens every day. Tightly balanced processes are necessary to ensure tolerance towards non-hazardous antigens, but to set up a powerful immune response against potentially dangerous ones. In this complex nexus of immune cells and their mediators, TRM cells play a central role and have been shown to promote both physiological and pathological events. In this review, we will summarize the current knowledge on the homeostatic functions of TRM cells and delineate their implication in infection control in the gut. Moreover, we will outline their commitment in immune dysregulation in gastrointestinal chronic inflammatory conditions and shed light on TRM cells as current and potential future therapeutic targets.

Development of equivalent chain length (ECL) rules for lipid compounds.

Lipid compounds (fatty acids, tocochromanols, phytosterols) are difficult to separate by countercurrent chromatography (CCC) due to the existence of many similar structures and the limited availability of suitable biphasic solvent systems. Here we show that for these compound classes the success of a CCC separation can be directly derived from the chemical structures without the necessity of experimental determinations of K values. In most cases, lipid compounds differ in the total carbon number and the number of double bonds. For each structure the so-called equivalent chain length (ECL) can be calculated by subtracting a distinct value for each double bond from the total carbon number. Empirically, we verified that in the case of unbranched fatty acids (determined as methyl esters) one double bond corresponds with two carbons. Evaluation of CCC data from seventeen phytosterols in five plant oils and nine tocochromanol standards showed that one double bond was equal with one carbon for both lipid classes. Most compounds with different ECL can be separated by CCC but not those with the same ECL. In these cases, the selection of a suitable source for isolation of a particular lipid compound becomes very important. Knowledge of the impact of double bonds may also be a helpful tool for other substance classes.

Nutritional Value of the Duckweed Species of the Genus Wolffia (Lemnaceae) as Human Food.

Species of the genus Wolffia are traditionally used as human food in some of the Asian countries. Therefore, all 11 species of this genus, identified by molecular barcoding, were investigated for ingredients relevant to human nutrition. The total protein content varied between 20 and 30% of the freeze-dry weight, the starch content between 10 and 20%, the fat content between 1 and 5%, and the fiber content was ~25%. The essential amino acid content was higher or close to the requirements of preschool-aged children according to standards of the World Health Organization. The fat content was low, but the fraction of polyunsaturated fatty acids was above 60% of total fat and the content of n-3 polyunsaturated fatty acids was higher than that of n-6 polyunsaturated fatty acids in most species. The content of macro- and microelements (minerals) not only depended on the cultivation conditions but also on the genetic background of the species. This holds true also for the content of tocopherols, several carotenoids and phytosterols in different species and even intraspecific, clonal differences were detected in Wolffia globosa and Wolffia arrhiza. Thus, the selection of suitable clones for further applications is important. Due to the very fast growth and the highest yield in most of the nutrients, Wolffia microscopica has a high potential for practical applications in human nutrition.