Dysregulated Macrophage Pro-Inflammatory Cytokine Production and Chronic Colitis in Mice Lacking Both Gab2 and Gab3

Macrophages are responsible for protecting the body against foreign invaders. We have been studying the role of Grb2-associated binding proteins (Gabs) in macrophage biology. In mice, Gabs are adaptor proteins that include three family members (Gab1, Gab2, and Gab3) that play critical regulatory roles in modulating cytokine receptor signaling. Gab2 knockout mice have no developmental defects but have impaired allergic responses, osteoclast defects, altered mast cell development, and altered hematopoiesis. Gab3 knockout mice have no defined phenotypes alone and although highly expressed in macrophages, a functional role was not found despite considerable focus on this cell type. Therefore, we set out to determine the combined role of Gab2 and Gab3 to determine whether they performed redundant functions not observable in single knockout mice. To analyze regulation of macrophage cytokine production, a Gab2/3 deficient mouse model was generated on the C57BL/6 background. Bone Marrow Derived Macrophages (BMDM) were expanded from the bone marrow (BM) of wild-type (WT), Gab2 and Gab3 single knockout and Gab2/3 knockout mice and found to similarly co-express CD11b and F4/80. However, Gab2/3 knockout BM produced only 30% of wild-type BMDM numbers. Despite reductions in BMDM absolute numbers, isolated BMDM demonstrated significant induction of pro-inflammatory cytokines TNF-α and IL-12 and anti-inflammatory cytokine IL-10 mRNA at baseline. Interestingly, after LPS stimulation (100ng/ml) we detected much greater induction of TNF-α and IL-12 mRNA and protein expression. Interestingly, despite increased IL-10 mRNA induction in Gab2/3 knockout BMDM, no IL-10 protein expression could be detected by Luminex assay. No changes were observed in production of interferon or STAT1 activation in these BMDM. Studies have shown that rapamycin treatment of macrophages suppresses mTORC1 and subsequently reduces IL-10 production and promotes pro-inflammatory cytokine production. Gab2 is known for its role in regulating the PI3K pathway through interactions with the p85 regulatory subunit of PI3K. Therefore, we also examined whether mTOR activation was effected by Gab2/3 deficiency causing altered cytokine expression. Deletion of Gab2/3 in BMDMs treated with LPS showed an inhibition of 4EBP1 phosphorylation and increased AKT phosphorylation. These results suggest that Gabs may play a critical role in modulating mTOR activation and potentially causing defects in protein translation that reflect in reduced IL-10 cytokine levels in Gab2/3 knockout cells. IL-10 has a critical immunoregulatory role that is dysregulated in patients with inflammatory bowel disease. IL-10 deficient mice develop colitis due to loss of mucosal immune tolerance. Strikingly, as early as two months of age in vivo 12/32 (37.5%) Gab2/3 knockout mice developed rectal prolapse and suffered from diarrhea within a six month period. Histological analysis of isolated colons using a scoring system confirmed spontaneous development of colitis in Gab2/3 knockout mice compared to no phenotypes observed in WT and single knockout controls. To determine whether the BM was directly involved in the disease, BM chimeras were generated using irradiated WT mice as recipients and Gab2/3 knockout mice as donors. Susceptible recipients receiving Gab2/3 knockout BM showed a more invasive colitis phenotype than the spontaneous disease and resulted in forced euthanization due to body weight decreases greater than 25%. Multiple ulcerations were present in most of the colon proximal region, with extensive epithelial damage, transmural inflammation, and in some mice adenocarcinoma. Notably, we did not observe adenocarcinoma in untransplanted Gab2/3 knockout mice, suggesting that epithelial deletion of Gab2/3 may suppress cancer whereas in the bone marrow chimera model, the epithelial cells are WT and can be transformed. Similar phenotypes were also observed in secondary transplant recipients. Lastly, treatment of Gab2/3 knockout mice with dextran-sodium-sulfate (DSS) induced rapid severe colitis that resulted in death of 80% and 40% of Gab2/3 knockout and WT mice respectively. Overall, these observations demonstrate a major redundant role for Gab2 and Gab3 in macrophage immune surveillance required for the prevention of colitis in mice.