Targeted Intestinal Tight Junction Hyperpermeability Alters the Microbiome, Behavior, and Visceromotor Responses.

Markedly increases in intestinal permeability occur in inflammatory bowel disease, graft-versus-host disease, celiac disease, and multiple organ dysfunction. In these diseases, effectors of increased permeability include immune signaling, microbiome, and corticosteroids that, in part, signal through epithelial myosin light chain kinase (MLCK). More modest permeability increases occur in other disorders, including irritable bowel syndrome (IBS), autism spectrum disorder, depression, and stress-related disorders. However, data directly linking barrier loss to phenotypes of these diseases are lacking.

To define the impact of modestly increased intestinal permeability, we studied transgenic mice with intestinal epithelial-specific constitutively-active myosin light chain kinase (CAMLCK) expression. This MLCK-dependent tight junction regulation increased intestinal permeability (Supplementary Figure S1A and B). Nevertheless, postnatal growth (Supplementary Figure S1C), reproduction, intestinal transit (Supplementary Figure S1D), intestinal histology, epithelial proliferation (a sensitive indicator of epithelial damage), and epithelial turnover are unaffected in CAMLCK transgenic (CAMLCK) mice. In contrast, mucosal tumor necrosis factor-α, interferon-γ, interleukin (IL)-10, and IL-13 transcripts as well as numbers of lamina propria neutrophils, CD4+ T cells, and IgA+ plasma cells are modestly increased by CAMLCK expression., Subclinical inflammation is, therefore, present and, by microbiome-dependent, IL-17–mediated processes, affords partial protection from acute pathogen invasion. Immune activation is nevertheless unlikely to amplify CAMLCK-driven permeability increases, as barrier function and ZO-1 anchoring are both acutely normalized by enzymatic MLCK inhibition.,

Supplementary Figure 1

(A) Transjejunal fluorescein flux was increased in CAMLCK (blue circles) relative to wild-type (WT) (red squares) littermates. Values are mean ± SD. ∗P < .05. Mann-Whitney U test. (B) In vivo analysis using FITC-4kDa dextran demonstrated increased permeability of CAMLCK (blue circles, n = 19) relative to WT (red squares, n = 20) littermates. Values are mean ± SD. ∗P < .05, t test. (C) Weight gain was similar in WT (red squares, n = 6) and CAMLCK (blue circles, n = 6) littermates. Values are mean ± SD. (D) Intestinal transit was similar in WT (red squares, n = 10) and CAMLCK (blue circles, n = 9) littermates. Values are mean ± SD. (E) Partial least-squares discriminant analysis (PLS-DA) score plot based on the relative abundances of 18 microbial taxa in gut contents of CAMLCK (circles, n = 16) and WT (squares, n = 15) born to 8 different dams (each color represents 1 dam). (F) Relative abundances of microbial communities in CAMLCK (blue) and WT (red) mice. Diagrams indicate regions analyzed.

We initially analyzed the gut microbiome of 31 wild-type (WT) and CAMLCK pups born to 8 WT dams. The microbiomes segregated by pup genotype but not dam (Supplementary Figure S1E) and included increased Clostridium and decreased Bacteroidetes, Enterococcus spp, and Prevotella in CAMLCK mice (Supplementary Figure S1F). Increased intestinal permeability can therefore cause dysbiosis-like microbiome shifts. Interestingly, maternal separation, which increases intestinal permeability, causes similar alterations and can be partially corrected by MLCK inhibitor–induced barrier restoration.

Microbiome alterations overlapping with the above have been reported in IBS and autism spectrum disorder. We therefore asked if CAMLCK mice displayed anxiety-like behavior, as occurs in those disorders, using the open field test (Figure 1A). Both the percentage of distance traveled in the center and the fraction of time spent in the center of the open field were reduced in CAMLCK mice (Figure 1A); this did not reflect reduced locomotor activity, as total distance traveled in the entire area was similar in CAMLCK and WT mice (Figure 1A). These data are consistent with increased anxiety-like behavior in CAMLCK mice. Although the results cannot differentiate between direct effects of increased permeability and those requiring intermediate mediators, these data demonstrate that intestinal permeability increases can trigger behavioral changes.

Figure 1

Increased intestinal permeability modifies behavior and visceral sensitivity. (A) Videotracking paths of representative WT and CAMLCK mice in the open field test. Percent distance traveled in the center (dashed lines), percent time in the center, and overall distance traveled in the entire field are shown. CAMLCK (blue circles, n = 8) and WT (red squares, n = 9) littermates were tested. Values are mean ± SEM. ∗P < .05; ∗∗P < .01, Mann-Whitney U test. (B) Stepwise colorectal distension-induced visceromotor responses in CAMLCK (blue circles, n = 7) were reduced relative to WT (red squares, n = 7) littermates. Genotype-specific differences were eliminated by MLCK inhibition, water avoidance stress, or naloxone treatment. n = 5–9 per condition; for each treatment (vehicle control CAMLCK and WT mice from the same experiment are shown with pale symbols in the last three graphs). Values are mean ± SEM. ∗∗, P < .01, 2-way analysis of variance.

Stress and increased permeability have been associated with enhanced visceral sensitivity in humans and rodents. Surprisingly, CAMLCK mice displayed striking visceral analgesia to colorectal distension relative to WT littermates (Figure 1B). Sensitivity was restored by enzymatic MLCK inhibition, water avoidance stress, or naloxone-mediated opioid receptor antagonism (Figure 1B). Although this effect of increased permeability on visceral sensitivity was unexpected, it is remarkably similar to the naloxone-reversible visceral analgesia reported in chronically stressed female rats and naloxone-sensitive inhibition of nociceptive neurons by supernatants of colitic human and murine tissues.

Studies of female IBS patients have linked increased permeability to altered functional and structural brain connectivity. Thus, although responses to colorectal distension can be mediated by spinal reflexes as well as sensory, limbic, and paralimbic regions of the brain, we asked if neuronal activation was modified by CAMLCK-induced permeability increases. C-Fos immunolabeling, an indicator of neuronal activity, was significantly greater in the paraventricular nucleus of the thalamus, the paraventricular nucleus of the hypothalamus, and the hippocampus but not the medial prefrontal cortex, nucleus accumbens, or amygdala of CAMLCK, relative to WT, mice (Figure 2, Supplementary Figure S2). Increased intestinal permeability may therefore increase basal neuronal activity in areas of the brain that regulate responses to visceral pain or stress but not those associated with conscious visceral sensation.

Figure 2

Increased intestinal permeability induces increased c-Fos immunolabeling in selected brain regions. CAMLCK (blue circles, n = 5–6) and WT (red squares, n = 5–6) littermates. Representative images of c-Fos–immunolabeled brains from CAMLCK and WT mice. Scale bars = 200 μm. Values are mean ± SD. ∗P < .05, t test.

Supplemental Figure 2

CAMLCK (blue circles, n = 5–6) and wild-type (WT) (red squares, n = 5–6) littermates. Representative images of c-Fos–immunolabeled brains from CAMLCK and WT mice. Scale bars = 200 μm. Values are mean ± SD. ∗P < .05, t test.

These results demonstrate that increased intestinal permeability can impact (1) gut microbiome composition, (2) behavior, (3) visceral pain responses, and (4) neuronal activation within the brain. Critically, these changes are all results, rather than causes, of intestinal barrier loss, as the latter was induced by targeted CAMLCK expression.

The sites of neuronal activation in CAMLCK mice support the hypothesis that increased intestinal permeability can activate the hypothalamic-pituitary-adrenal axis. Conversely, hypothalamic-pituitary-adrenal axis activation by exogenous stress can induce intestinal permeability increases. Thus, as has been proposed in inflammatory bowel disease and graft-versus-host disease, a self-amplifying cycle may ultimately direct the diverse phenotypes induced by MLCK-dependent, intestinal permeability increases. Further study is needed to define the complex relationships between intestinal permeability, stress, behavioral alterations, visceromotor responses, microbiome composition, and other abnormalities.

These data are the first to assess behavior in a model in which a targeted increase in intestinal tight junction permeability is the only direct perturbation. The results demonstrate, unequivocally, that modest tight junction permeability increases induced via a physiologically and pathophysiologically relevant mechanism are sufficient to trigger local and systemic microbial, behavioral, and neurosensory changes. This provides a new perspective with which to understand previously hypothesized cause-effect relationships that have been proposed on the basis of correlative data.