Inactivation of paracellular cation-selective claudin-2 channels attenuates immune-mediated experimental colitis in mice

Abstract

the larger cations tetramethylammonium, tetraethylammonium, or N-methyl-D-glucamine (Figure 1E). This size-selective permeability increase is characteristic of claudin-2 channel activity (14-16, 48-50), but does not exclude the possibility that other cation-selective pore-forming claudins, e.g., claudin-15 (10, 13, 22), contribute to IL-13-induced barrier loss in vivo. We therefore asked if claudin-2 was required for IL-13-induced barrier loss. Prior to IL-13 treatment, paracellular permeability was similar in Cldn2 +/+ and Cldn2-/mice, consistent with limited claudin-2 expression in adults. IL-13 treatment was not, however, able to increase paracellular permeability of small cations, including Na + and methylamine in Cldn2-/mice (Figure 1F). Claudin-2 is therefore necessary for IL-13-induced increases in intestinal pore pathway permeability. In order to determine if claudin-2 upregulation is sufficient to mediate the effects of IL-13 on intestinal cation permeability, transgenic mice expressing GFP-tagged mouse claudin-2 from the intestinal epithelium-specific villin (Vil1) promoter (Cldn2 Tg) were characterized (29). Total claudin-2 expression (the sum of endogenous and GFP-claudin-2) in these Cldn2 Tg mice was similar in distribution (Figure 1G) to that of IL-13-treated Cldn2 +/+ mice (Figure 1B) and increased 3.3-fold relative to Cldn2 +/+ mice that did not receive IL-13 (Figure 1, H and I). Moreover, transgenic GFPclaudin-2 expression selectively increased paracellular permeability of Na + , methylamine, and, to a lesser extent, ethylamine, in a manner that recapitulated the effect of IL-13 on Cldn2 +/+ mice (Figure 1J). Claudin-2 is, therefore, both necessary and sufficient to mediate IL-13-induced barrier loss in vivo. Transgenic claudin-2 expression exacerbates immune-mediated colitis. We have found that severity and duration of infectious colitis are reduced in these GFP-Cldn2 Tg mice (29). A study of similar transgenic mice expressing human claudin-2 from the same Vil1 promoter demonstrated that claudin-2 overexpression was also protective in DSS-induced, chemical colitis (41). Conversely, claudin-2 knockout exacerbated infectious and chemical colitis (29, 42). These data suggest that claudin-2-mediated pore pathway permeability increases may be adaptive, i.e., beneficial in IBD. To test this hypothesis, we compared severity of experimental IBD in immunodeficient Cldn2 Tg Rag1-/and Cldn2 +/+ Rag1-/mice using the well-established T cell transfer model (33, 51). Unexpectedly, disease was significantly more severe in Cldn2 Tg Rag1-/mice relative to Cldn2 +/+ Rag1-/mice. This was demonstrated by more extensive weight loss (Figure 2A) and greater disease activity (Figure 2B). Consistent with increased disease severity, leak (macromolecular) and unrestricted (epithelial damage) pathway permeabilities, measured as 4-kDa and 70-kDa dextran flux, respectively, were markedly greater in Cldn2 Tg Rag1-/mice relative to Cldn2 +/+ Rag1-/mice (Figure 2C and ref. 29). Colitis was associated with increased claudin-2 exp...

Doxorubicin increases permeability of murine small intestinal epithelium and cultured T84 monolayers

“…Recent work by Raju et al has shown that increased expression of Claudin-2 enhances immune mediated colitis specifically via the pore pathway and not via the leak pathway; however, they also showed that Cldn2 −/− were protected from the immune mediated increase in the leak pathway 34 . The pleiotropic nature of tight junctional proteins, specifically Claudin-2, suggests that changes in expression may have effects on both the leak and pore pathway 35 .…”

Culture of Intestinal Epithelial Cell Monolayers and Their Use in Multiplex Macromolecular Permeability Assays for In Vitro Analysis of Tight Junction Size Selectivity

“…Intestinal epithelial claudin‐2 expression can be upregulated in vivo by IL‐13 or IL‐22 as well as enteric infection and immune‐mediated disease (Epple et al., 2017; Heller et al., 2005; Ong et al., 2020; Raju et al., 2020; Schumann et al., 2012; Tsai et al., 2017). In Citrobacter rodentium infection, IL‐22‐induced claudin‐2 upregulation increases paracellular permeability to creatinine (6 Å diameter), but not 4 or 70 kDa dextran (28 and 120 Å diameter), within 2 days of infection.…”

“…Studies of knockout mice lacking claudin‐2 and transgenic mice overexpressing intestinal epithelial‐specific claudin‐2 have shown that this claudin‐2‐dependent permeability promotes pathogen clearance and is protective in C. rodentium infection (Tsai et al., 2017). Conversely, claudin‐2 upregulation in immune‐mediated experimental inflammatory bowel disease promotes disease progression by poorly understood mechanisms (Raju et al., 2020).…”

“…Many cytokines also reduce barrier function via a tight junction–independent, non‐selective route, the unrestricted pathway, at sites of epithelial damage (Fig. 1; Marchiando et al., 2011; Moore, Carlson, & Madara, 1989; Nalle & Turner, 2015; Nusrat, Delp, & Madara, 1992; Raju et al., 2020; Su et al., 2013; Tsai et al., 2017).…”

“…1; Turner, 2009). Some cytokines, including IL‐13 and IL‐22, specifically increase pore pathway permeability (Raju et al., 2020; Tsai et al., 2017; Van Itallie et al., 2008; Wang, Mumm, Herbst, Kolbeck, & Wang, 2017; Weber et al., 2010). In contrast, other inflammatory mediators, such as TNF, LIGHT, and IL‐1β, increase paracellular permeability of the low‐capacity, charge‐nonselective leak pathway that mediates flux of macromolecules up to ∼100 Å diameter (Al‐Sadi et al., 2013; Al‐Sadi, Ye, Dokladny, & Ma, 2008; Bruewer et al., 2003; Clayburgh et al., 2005; Clayburgh, Musch, Leitges, Fu, & Turner, 2006; Schwarz et al., 2007; Wang et al., 2005; Weber et al., 2010; Zolotarevsky et al., 2002).…”