Attenuation of NF-κB in Intestinal Epithelial Cells Is Sufficient to Mitigate the Bone Loss Comorbidity of Experimental Mouse Colitis

Abstract

Skeletal abnormalities are common comorbidities of inflammatory bowel disease (IBD). Patients suffering from IBD, including ulcerative colitis and Crohn's disease, present with skeletal complications. However, the mechanism underpinning IBD-associated bone loss remains vague. Intestinal inflammation generates an inflammatory milieu at the intestinal epithelium that leads to dysregulation of mucosal immunity through gut-residing innate lymphoid cells (ILCs) and other cell types. ILCs are recently identified mucosal cells considered as the gatekeeper of gut immunity and their function is regulated by intestinal epithelial cell (IEC)-secreted cytokines in response to the inflammatory microenvironment. We first demonstrate that serum as well as IECs collected from the intestine of dextran sulfate sodium (DSS)-induced colitis mice contain high levels of inflammatory and osteoclastogenic cytokines. Mechanistically, heightened inflammatory response of IECs was associated with significant intrinsic activation of NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) in IECs and increased frequency of ILC1, ILC3, and myeloid osteoclast progenitors. Validating the central role of IEC-specific NF-κB activation in this phenomenon, conditional expression of constitutively active inhibitor kappa B kinase 2 (IKK2) in IECs in mice recapitulates the majority of the cellular, inflammatory, and osteolytic phenotypes observed in the chemically induced colitis. Furthermore, conditional deletion of IKK2 from IECs significantly attenuated inflammation and bone loss in DSS-induced colitis. Finally, using the DSS-induced colitis model, pharmacologic inhibition of IKK2 was effective in reducing frequency of ILC1 and ILC3 cells, attenuated circulating levels of inflammatory cytokines, and halted colitis-associated bone loss. Our findings identify IKK2 in IECs as viable therapeutic target for colitis-associated osteopenia. Statistical analysisResults are presented as 1) mean ± SD; 2) fold change; and 3) representative images. Sample size ranged from 5 to 12 animals per group based on specific experiments. In cases where sufficient numbers of transgenic mice were not available ◼ 2 KE ET AL.

Mechanisms Underlying Bone Loss Associated with Gut Inflammation

“…Typically, proinflammatory cytokines TNFα and IL-1β, which are increased in IBD patients, have been shown to directly increase IEC permeability by interrupting TJ proteins, associated with increased activation of NF-κB signaling, the primary inflammatory response pathway [159][160][161][162]. Those results are consistent with our findings in IKK2ca IEC mice, wherein constitutive activation of NF-κB signaling pathway in IECs [80], which mimics chronic inflammation, can induce gut damage and directly affect bone regulating cells (such as OC precursors) via gut-secreted cytokines.…”

“…A similar therapeutic function of OPG in gut-inflammation associated bone loss was also found in IL-2−/− colitis mice, in which both skeletal abnormalities and colitis score were reduced by modulation of RANKL-RANK interactions with exogenous administration of Fc-OPG [72]. The potential critical effect of systemic and intestinal RANKL in bone loss and osteoclastogenesis is also revealed in our recent study [80]. To sum up, the role of the RANK/RANKL signaling pathway in location inflammation is still needed to be addressed in different gut inflammation models.…”

“…To further interrogate the role of NF-kB signaling in the gut-bone axis, our lab generated an IEC-specific model of NF-κB overactivation. The findings from our studies using IEC-specific constitutively activated IKK2 mice (IKK2ca IEC ) model highlight direct and crucial role of intestinal NF-κB signaling in linking chronic gut inflammation with bone loss [80]. IKK2ca IEC develop a mild inflammation in small intestine [81]; however, with significantly elevated serum and epithelial level of inflammatory cytokines.…”

“…In addition, IgA antibodies found in breast milk have been shown to improve gut microbiota and overall intestinal health, reducing the risk of IBD. Although more evidence is needed to prove an association between bone loss and intestinal barrier dysfunction, studies on animal models using chemically-induced barrier permeability [93,149] and by targeting epithelial function through genetic modification [80], support this notion. A major drawback of this clinical finding is small sample sizes and highly specific patient populations that may not represent broad patient groups.…”

“…These cells produce high quantities of pro-inflammatory cytokines (IL-1β and TNFα), which are more actively produced at the onset of gut inflammation [193,194]. Our most recent studies showed both IEC and ILCs, the representative innate immunity regulating components, are responsible for initiation of murine osteopenia due to intestinal barrier dysfunction [80]. Furthermore, since IBD is characterized by recurrent chronic inflammation, the adaptive immune response plays a key role in the development of IBD-associated bone loss [195].…”