IL-17A Produced by Innate Lymphoid Cells Is Essential for Intestinal Ischemia-Reperfusion Injury.

Ischemia-reperfusion (IR) injury to the small intestine following clamping of the superior mesenteric artery results in an intense local inflammatory response that is characterized by villous damage and neutrophil infiltration. IL-17A, a cytokine produced by a variety of cells in response to inflammatory cytokines released following tissue injury, has been implicated in IR injury. Using Il17a-/- , Il23r-/- , and Rorc-/- mice and administration of anti-IL-17A and anti-IL-23 neutralizing Abs to wild-type mice, we demonstrate that intestinal IR injury depends on IL-17A and that IL-17A is downstream of the binding of autoantibody to ischemia-conditioned tissues and subsequent complement activation. Using bone marrow chimeras, we demonstrate that the IL-17A required for intestinal IR injury is derived from hematopoietic cells. Finally, by transferring autoantibody-rich sera into Rag2γc-/- and Rag2-/- mice, we demonstrate that innate lymphoid cells are the main producers of IL-17A in intestinal IR injury. We propose that local production of IL-17A by innate lymphoid cells is crucial for the development of intestinal IR injury and may provide a therapeutic target for clinical exploitation.

Intracellular Activation of Complement 3 Is Responsible for Intestinal Tissue Damage during Mesenteric Ischemia.

Intestinal ischemia followed by reperfusion leads to local and remote organ injury attributed to inflammatory response during the reperfusion phase. The extent to which ischemia contributes to ischemia/reperfusion injury has not been thoroughly studied. After careful evaluation of intestinal tissue following 30 min of ischemia, we noticed significant local mucosal injury in wild-type mice. This injury was drastically reduced in C3-deficient mice, suggesting C3 involvement. Depletion of circulating complement with cobra venom factor eliminated, as expected, injury recorded at the end of the reperfusion phase but failed to eliminate injury that occurred during the ischemic phase. Immunohistochemical studies showed that tissue damage during ischemia was associated with increased expression of C3/C3 fragments primarily in the intestinal epithelial cells, suggesting local involvement of complement. In vitro studies using Caco2 intestinal epithelial cells showed that in the presence of LPS or exposure to hypoxic conditions the cells produce higher C3 mRNA as well as C3a fragment. Caco2 cells were also noted to produce cathepsins B and L, and inhibition of cathepsins suppressed the release of C3a. Finally, we found that mice treated with a cathepsin inhibitor and cathepsin B-deficient mice suffer limited intestinal injury during the ischemic phase. To our knowledge, our findings demonstrate for the first time that significant intestinal injury occurs during ischemia prior to reperfusion and that this is due to activation of C3 within the intestinal epithelial cells in a cathepsin-dependent manner. Modulation of cathepsin activity may prevent injury of organs exposed to ischemia.

An ultrasensitive assay format for detecting ULK1 inhibition by monitoring the phosphorylation status of Atg13.

A new technology from Quanterix called SiMoA (single molecule array) which employs a fully automated system capable of ultrasensitive sandwich based ELISA detection was explored. Our studies focused upon the inhibition of the autophagy initiating kinase ULK1 by measuring the both total Atg13 and the phosphorylation of Atg13(pSer(318)) from control and following compound treatment in either overexpressing or wild type tissue culture samples. The results show linear protein concentration dependence over two orders of magnitude and provide an assay window of 8- to 100-fold signal to background for inhibition of phosphorylation for both wild type and overexpressed samples, respectively. Moreover, overexpressed samples displayed 17-fold pSer(318)-Atg13 above wild type levels of with no apparent differences in compound potency. Lastly, the inhibition of ULK1 from mouse derived wild type xenografts also demonstrated loss of pSer(318)-Atg13 upon ULK1 inhibitor treatment that compared favorably to Western blot. These results show that the SiMoA technology can detect quantitatively low levels of endogenous biomarkers with the ability to detect the loss of pSer(318)-Atg13 upon ULK1 inhibition.

Complement receptor of the immunoglobulin superfamily reduces murine lupus nephritis and cutaneous disease.

Complement activation takes place in autoimmune diseases and accounts for tissue inflammation. Previously, complement inhibition has been considered for the treatment of SLE. Complement receptor of the immunoglobulin superfamily (CRIg) is a selective inhibitor of the alternative pathway of complement and a soluble form reverses established inflammation and bone destruction in experimental autoimmune arthritis. We asked whether specific inhibition of the alternative pathway could inhibit autoimmunity and/or organ damage in lupus-prone mice. Accordingly, we treated lupus-prone MRL/lpr mice with a soluble form of CRIg (CRIg-Fc) and we found that it significantly diminished skin lesions, proteinuria and pyuria, and kidney pathology. Interestingly, serum levels of anti-DNA antibodies were not affected despite the fact that serum complement 3 (C3) levels increased significantly. Immunofluorescent staining of kidney tissues revealed a reduction in staining intensity for C3, IgG, and the macrophage marker Mac-2. Thus our data show that inhibition of the alternative pathway of complement controls skin and kidney inflammation even in the absence of an effect on the production of autoantibodies. We propose that CRIg should be considered for clinical trials in patients with systemic lupus erythematosus.