Microbial Butyrate Synthesis Indicates Therapeutic Efficacy of Azathioprine in IBD Patients.

BACKGROUND AND AIMS: The microbial ecosystem seems to be an important player for therapeutic intervenption in inflammatory bowel disease [IBD]. We assessed longitudinal microbiome changes in IBD patients undergoing therapy with either azathioprine [AZA] or anti-tumour necrosis factor [anti-TNF] antibodies. We predicted the metabolic microbial community exchange and linked it to clinical outcome. METHODS: Faecal and blood samples were collected from 65 IBD patients at baseline and after 12 and 30 weeks on therapy. Clinical remission was defined as Crohn's Disease Activity Index [CDAI] < 150 in Crohn´s disease [CD], partial Mayo score <2 in ulcerative colitis [UC], and faecal calprotectin values <150 µg/g and C-reactive protein <5 mg/dl. 16S rRNA amplicon sequencing was performed. To predict microbial community metabolic processes, we constructed multispecies genome-scale metabolic network models. RESULTS: Paired Bray-Curtis distance between baseline and follow-up time points was significantly different for UC patients treated with anti-TNF antibodies. Longitudinal changes in taxa composition at phylum level showed a significant decrease of Proteobacteria and an increase of Bacteroidetes in CD patients responding to both therapies. At family level, Lactobacilli were associated with persistent disease and Bacteroides abundance with remission in CD. In-silico simulations of microbial metabolite exchange predicted a 1.7-fold higher butyrate production capacity of patients in remission compared with patients without remission [p = 0.041]. In this model, the difference in butyrate production between patients in remission and patients without remission was most pronounced in the CD group treated with AZA [p = 0.008]. CONCLUSIONS: In-silico simulation identifies microbial butyrate synthesis predictive of therapeutic efficacy in IBD.

Targeting Pyk2 to beta 1-integrin-containing focal contacts rescues fibronectin-stimulated signaling and haptotactic motility defects of focal adhesion kinase-null cells.

Focal adhesion kinase-null (FAK(-/-) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK(-/-) cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK(-/-) cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a beta 1-integrin-containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK(-/-) cells, exhibit elevated FN-stimulated extracellular signal-regulated kinase 2 (ERK2) and c-Jun NH(2)-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH(2)-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to beta-integrin-containing focal contact sites via interactions mediated by the FAK-CT domain.