Colonic Inhibition of Phosphatase and Tensin Homolog Increases Colitogenic Bacteria, Causing Development of Colitis in Il10-/- Mice.

Background: Phosphatase and tensin homolog (Pten) is capable of mediating microbe-induced immune responses in the gut. Thus, Pten deficiency in the intestine accelerates colitis development in Il10-/- mice. As some ambient pollutants inhibit Pten function and exposure to ambient pollutants may increase inflammatory bowel disease (IBD) incidence, it is of interest to examine how Pten inhibition could affect colitis development in genetically susceptible hosts. Methods: With human colonic mucosa biopsies from pediatric ulcerative colitis and non-IBD control subjects, we assessed the mRNA levels of the PTEN gene and the gene involved in IL10 responses. The data from the human tissues were corroborated by treating Il10-/-, Il10rb-/-, and wild-type C57BL/6 mice with Pten-specific inhibitor VO-OHpic. We evaluated the severity of mouse colitis by investigating the tissue histology and cytokine production. The gut microbiome was investigated by analyzing the 16S ribosomal RNA gene sequence with mouse fecal samples. Results: PTEN and IL10RB mRNA levels were reduced in the human colonic mucosa of pediatric ulcerative colitis compared with non-IBD subjects. Intracolonic treatment of the Pten inhibitor induced colitis in Il10-/- mice, characterized by reduced body weight, marked colonic damage, and increased production of inflammatory cytokines, whereas Il10rb-/- and wild-type C57BL/6 mice treated with the inhibitor did not develop colitis. Pten inhibitor treatment changed the fecal microbiome, with increased abundance of colitogenic bacteria Bacteroides and Akkermansia in Il10-/- mice. Conclusions: Loss of Pten function increases the levels of colitogenic bacteria in the gut, thereby inducing deleterious colitis in an Il10-deficient condition.

Src family kinase tyrosine phosphorylates Toll-like receptor 4 to dissociate MyD88 and Mal/Tirap, suppressing LPS-induced inflammatory responses.

Src family kinases (SFKs) are a family of protein tyrosine kinases containing nine members: Src, Lyn, Fgr, Hck, Lck, Fyn, Blk, Yes, and Ylk. Although SFK activation is a major immediate signaling event in LPS/Toll-like receptor 4 (TLR4) signaling, its precise role has remained elusive due to various contradictory results obtained from a certain SFK member-deficient mice or cells. The observed inconsistencies may be due to the compensation or redundancy by other SFKs upon a SFK deficiency. The chemical rescuing approach was suggested to induce temporal and precise SFK activation in living cells, thereby limiting the chance of cellular adaption to a SFK-deficient condition. Using the rescuing approach, we demonstrate that restoring SFK activity not only induces tyrosine phosphorylation of TLR4, but also inhibits LPS-induced NFκB and JNK1/2 activation and consequently suppresses LPS-induced cytokine production. TLR4 normally recruits TIR domain-containing adaptors in response to LPS, however, temporally restored SFK activation disrupts the LPS-induced association of MyD88 and Mal/Tirap with TLR4. Additionally, using kinase-dead SFK-Lyn (Y397/508F) and constitutively active SFK-Lyn (Y508F), we found that the kinase-dead SFK inhibits TLR4 tyrosine phosphorylation with reduced binding affinity to TLR4, while the kinase-active SFK strongly binds to TLR4 and promotes TLR4 tyrosine phosphorylation, suggesting that SFK kinase activity is required for TLR4 tyrosine phosphorylation and TLR4-SFK interaction. Together, our results demonstrate that SFK activation induces TLR4 tyrosine phosphorylation, consequently dissociating MyD88 and Mal/Tirap from TLR4 and inhibiting LPS-induced inflammatory responses, suggesting a negative feedback loop regulated by SFK-induced tyrosine phosphorylation in TLR4.