# Upregulation of TCPTP in Macrophages Is Involved in IL-35 Mediated Attenuation of Experimental Colitis

## TL;DR

This study shows that IL-35 reduces colitis severity by increasing TCPTP in macrophages, which helps control inflammation.

## Contribution

The novel finding is that TCPTP upregulation in macrophages is a key mechanism by which IL-35 attenuates colitis.

## Key findings

- Pichia/IL-35 treatment improved weight loss, bloody stools, and colon shortening in a colitis model.
- IL-35 increased M2 macrophages and Tregs while decreasing M1 macrophages, Th17, Th1 cells, and IL-6.
- IL-35 promotes TCPTP expression in macrophages both in vitro and in vivo.

## Abstract

Ulcerative colitis (UC) is a chronic intestinal inflammatory disease with complex etiology. Interleukin-35 (IL-35), as a cytokine with immunomodulatory function, has been shown to have therapeutic effects on UC, but its mechanism is not yet clear. Therefore, we constructed Pichia pastoris stably expressing IL-35 which enables the cytokines to reach the diseased mucosa, and explored whether upregulation of T-cell protein tyrosine phosphatase (TCPTP) in macrophages is involved in the mechanisms of IL-35-mediated attenuation of UC. After the successful construction of engineered bacteria expressing IL-35, a colitis model was successfully induced by giving BALB/c mice a solution containing 3% dextran sulfate sodium (DSS). Mice were treated with Pichia/IL-35, empty plasmid-transformed Pichia (Pichia/0), or PBS by gavage, respectively. The expression of TCPTP in macrophages (RAW264.7, BMDMs) and intestinal tissues after IL-35 treatment was detected. After administration of Pichia/IL-35, the mice showed significant improvement in weight loss, bloody stools, and shortened colon. Colon pathology also showed that the inflammatory condition of mice in the Pichia/IL-35 treatment group was alleviated. Notably, Pichia/IL-35 treatment not only increases local M2 macrophages but also decreases the expression of inflammatory cytokine IL-6 in the colon. With Pichia/IL-35 treatment, the proportion of M1 macrophages, Th17, and Th1 cells in mouse MLNs were markedly decreased, while Tregs were significantly increased. In vitro experiments, IL-35 significantly promoted the expression of TCPTP in macrophages stimulated with LPS. Similarly, the mice in the Pichia/IL-35 group also expressed more TCPTP than that of the untreated group and the Pichia/0 group.