NF-κB/HIF-1α Pathway Involved In IFN-γ Induced Intestinal Epithelial Barrier Disfunction

BackgroundThe intestinal epithelial barrier (IEB) is most important barrier against the externalenvironment. It acts as a selectively permeable barrier, permitting the absorption of nutrients,electrolytes, and water while maintaining an effective defense against intraluminal antigens,pathogens and toxins entering the mucosal tissues and potentially causing disease. Thisdefensive system consists of mechanical barrier, immune barrier, biological barrier andchemical barrier, in which, mechanical barrier is the most important structure foundation inmaintaining intestinal barrier function. The damage of this structural can lead to intestinalbarrier dysfunction, which induced bacterial translocation, enterogenic infection,aggravating the primary disease, systemic inflammatory response syndrome (SIRS), andeven multiple organ dysfunction syndrome (MODS). Interferon-, one of the importantinflammatory mediators induced by intestinal intraepithelial iymphocytes (iIEL), playing animportant role in intestinal barrier dysfunction, while the precise mechanisms involved arestill unclear.Hypoxia-inducible factor-1(HIF-1), a transcription factor induced in hypoxiacondition, has emerged as a central determinant in the pathophysiological response tocellular hypoxia. HIF-1plays key roles in energy metabolism, ion metabolism,catecholamine metabolism, angiogenesis, vasoconstriction control and apoptosis of tumorcells. Recent studies have showed that HIF-1α activation is deleterious to intestinal barrierfunction associated with hypoxia, ischemia/reperfusion, and inflammation.Recently, induction of HIF-1α by proinflammatory cytokines, not only under hypoxiabut also under normoxic conditions, has been described in numbers of studies. Many of thestimuli that induce HIF-1α in normoxia are known to activate a number of othertranscription factors such as NF-κB. Rescent study has showed that IFN-γ mediated thetranscriptional regulation of HIF-1α in different ways, and activated the NF-κBpathway through a STAT1-independent pathway. Since IFN-γ and HIF-1α play important roles inintestinal barrier dysfunction, and NF-κBcould be activated by IFN-γ, the relationshipbetween these two factors in this process is still unknown.Therefore, in this study we investigated the role of NF-κB/HIF-1α pathway inintestinal epithelial barrier dysfunction induced by the IFN-γ. This study would be helpfulto provide a new theoretical basis for the prevention of the loss of intestinal epithelialbarrier function.Methods1. Intestinal epithelial cells (T84cells) were treated with different time of recombinantIFN-γ or different concentrations of recombinant IFN-γ or1%oxygen, the expression ofHIF-1α and HIF-1were detected by qRT-PCR,Western blot and confocal microscopymethod.2. T84cells were treated with IFN-γ or NF-κBpathway inhibitor PDTC, nuclear andcytoplasmic epithelial cell fractions were prepared to detect the activation of NF-κB.3. The effects of NF-κBinhibitor PDTC and HIF-1α inhibitor YC-1on the expressionof HIF-1α, tight junction proteins and morphological alteration of tight junction proteinswere investigated in T84cells treated with IFN-γ. The alteration of the transepithelialresistance (TER) was also mesured using a Millicell-ERS II system.Results1. Both hypoxia and IFN-γ treatment can lead to a dramatic increase of HIF-1a proteinexpression. The expression of HIF-1α protein was also induced in time-and dose-dependentmanners by IFN-γ, but IFN-γ did not induce any changes of HIF-1expression in a time-orconcentration-dependent manner.2. Treatment with IFN-γ not only induced the increase expression of HIF-1α, but alsoinduced nuclear translocation of HIF-1α, which indicated the activation of HIF-1.3. IFN-γ induced the activation of NF-κB, whereas pretreatment with PDTC not onlyinhibited IFN-γ-induced activation of NF-κB, but also inhibited the increase expression ofHIF-1α.4. Treatment with IFN-γ caused a decrease of TER by the time-and dose-dependentmanners. Both NF-κBinhibitor PDTC and HIF-1α inhibitor YC-1could inhibit theIFN-γ-induced drop in T84TER. 5. IFN-γ caused significant decrease in the expression of tight junction proteins ZO-1and Claudin-1, while increased the expression of Claudin-2. T84cells treated with IFN-γalso lead to a dramatic disruption of tight junctions in morphological alteration. Both NF-κBinhibitor PDTC and HIF-1α inhibitor YC-1attenuated this changes induced by IFN-γ.ConclusionsThis study suggested that IFN-γ induced the loss of epithelial barrier function anddisruption of tight junction proteins, by upregulation of HIF-1α expression through NF-κBpathway. The inhibition of NF-κBand HIF-1α pathways attenuate intestinal epithelialbarrier dysfunction caused by IFN-γ and prevent IFN-γ-induced morphological disruptionof tight junction in intestinal epithelia. Taken together, this study provides new insight intothe intracellular pathways that mediate the IFN-γ-induced disturbance of intestinal epithelialbarrier function.