3Ai–iii). IL-10 slightly induces Cldn1 mRNA in thio-PEM, but it rather suppressed the expression of this gene in BMDM. Of importance, the classical macrophage activators IFN-γ and LPS did not influence basal claudin-1 mRNA levels, except in C57BL/6 thio-PEM where IFN-γ slightly induces its expression. Finally, given the significant Cldn1 inducibility by TGF-β, we evaluated the presence of claudin-1 protein in TGF-β-stimulated

BALB/c thio-PEM. However, no claudin-1 could be detected by Western blot. Overall, Cldn1 gene expression AZD1208 seems to be predominantly regulated by TGF-β in macrophages. Stimulation of BALB/c thio-PEM with various cytokine combinations indicates that Cldn2 gene expression is induced by a variety of stimuli. While IL-4 induces check details claudin-2 mRNA levels 2.5-fold, IL-10 and TGF-β are slightly more effective inducers of claudin-2 mRNA, reaching a nearly 4-fold induction (Fig. 3Bi). The classical macrophage activators IFN-γ and LPS induced claudin-2 expression only faintly in these macrophages. Hence, in BALB/c thio-PEM, Cldn2 expression seems to be rather associated with alternative activation of macrophages. However, in C57BL/6 thio-PEM and BALB/c BMDM, almost all M2 and M1 stimuli induce Cldn2 gene transcription (Fig. 3Bii, iii). Collectively, Cldn2 gene expression does not discriminate between alternative or classical macrophage activation. Claudin-11 gene expression is

significantly induced in BALB/c thio-PEM by IL-4 and to a lesser extent also by IL-10 and TGF-β (Fig. 3Ci). The identification of IL-4 as most potent Cldn11 inducer can be extrapolated to C57BL/6 thio-PEM and especially BALB/c BMDM, in which we observed an 800-fold increase in claudin-11 transcripts upon IL-4 treatment. In both thio-PEM and BMDM, also IL-10 induces Cldn11, albeit at a lower level compared to IL-4 (Fig. 3Cii, iii). Importantly, IFN-γ 17-DMAG (Alvespimycin) HCl and LPS did not affect Cldn11 expression levels. Hence, claudin-11 behaves as a marker gene for AAMs in mouse macrophages.

In view of their in vitro induction by IL-4, we investigated whether Cldn1, Cldn2 and Cldn11 are also upregulated in macrophages during an IL-4-driven infectious disease in vivo. T. crassiceps helminths typically induce IL-4-dependent alternative macrophage activation during the chronic stage of infection [8]. Peritoneal macrophages isolated from 8-week infected mice expressed higher levels of Cldn1, Cldn2 and in particular Cldn11 transcripts, indeed illustrating the association of these genes with AAMs in vivo (Fig. 4A). Experimental infections with T. congolense parasites were documented to result in a switch from an inflammatory cytokine environment in the early phase of infection to an anti-inflammatory environment in the chronic stage. Correlating with this switch, splenic macrophages isolated during the early versus the chronic infection phase tend to be more M1 and M2, respectively.