The Role Of NOD2Signaling Pathway In Acute Lung Injury After Hemorrbagic Shock/Resuscitation

‘Second hit’ is used to describe the phenomenon that body reaction to infections isenhanced after hemorrhagic shock/resuscitation (HS/R), with slight infection beingable to cause systemic inflammation and multiple organ dysfunctions. As an openorgan, lung is susceptible to acute lung injury (ALI) during this HS/R process. Ourpreliminary data show that high-mobility group box1(HMGB1) increases theexpression of TLR2in alveolar macrophages (AM) by binding to TLR4after HS/R,and thereafter plays an important role in mediating ALI. Based on these findings andthe knowledge that NOD2, an intracellular pattern recognition receptor, is closelyassociated with inflammation, autophagy and apoptosis, we come up with ourhypothesis: HS/R may activate NOD2and MDP-NOD2signal, a ‘second hit’, mayaffect ALI after HS/R.Part1. The effect of HS/R on NOD2expression in AMTo study the effect of HS/R on NOD2expression in AM, we employed an HS/Rmodel in mice. We found that NOD2expression increased significantly in WT, but notTLR4-/-, mice, indicating that HS/R activates NOD2secondarily by binding to TLR4.Administration of neutralizing anti-HMGB1prior to HS/R prevented the upregulationof NOD2. These data demonstrated HS/R upregulates NOD2expression throughHMGB1-TLR4signaling pathway in AM. In the in vitro experiment, cultured WTAM were stimulated with HMGB1. We found that NOD2expression upregulationwas induced by HMGB1stimulation. Part2. The effect of NOD2on inflammatory response early after the‘second hit’To determine the effect of NOD2on lung inflammation after HS/R, we usedHS/R+MDP injection, a ‘second hit’ model mimicking clinical situations. Datashowed that the expression of MIP2, MIF and TNF-α was significantly increased,indicating that the upregulation of NOD2expression in AM after ‘second hit’mediated ALI. In addition, after performing the ‘second hit’ model in WT, TLR4-/-,NOD2-/-and NF-κB-/-mice, we examined the content of IL-1β in bronchoalveolarlavage (BAL) fluid. We found that TLR4, NOD2and NF-κB mediated the release ofIL-1β and also mediated lung inflammation both directly and indirectly. We alsostimulate AM sequentially with HMGB1and MDP, mimicking in vivo ‘second hit’model, to measure the activation of caspase-1and maturation of IL-1β. We foundthat HS/R mediated NOD2upregulation and thus increase the sensitivity of NOD2toMDP through HMGB1-TLR4signaling pathway. NOD2was also shown tomodulate IL-1β maturation and release and thus promote the release ofproinflammatory factors by regulating the activity of caspase-1.Part3. The role of NOD2in autophagosome formation during the‘second hit’To demonstrate whether MDP induces autophagy in macrophages, WT and NOD2-/-mice were treated with HS/R operation, then harvest the AM and stimulate them withMDP. T he cells were examined with immunofluorescence and Westren Blot for LC3protein. We found that HS/R could promote autophagy, which was caused by MDPadministration and could be blocked by3-MA. We also found that upregulation ofNOD2by HS/R promoted autophagy in AM in a HS/R+MDP ‘second hit’ model withWT and NOD2-/-mice. To examine the effect of HMGB1and MDP on autophagy inAM, we cultured mouse AM and stimulate them with HMGB1and MDP. LC3inAM was measured. We found that HMGB1upregulated NOD2expression in AMand then induced autophagy together with MDP. Part4. The role of NOD2-autophagy in ALI after the ‘second hit’We looked into the overall effect of NOD2-autophagy on animal survival after ‘secondhit’ with MDP. All HS/R WT mice survived for36h.75%or12.5%WT micesurvived when treated with an ‘second hit’ or3-MA+‘second hit’, respectively.However, all NOD2-/-mice survived regardless of MDP or3-MA administration. Toinvestigate the effect of NOD2-autophagy signaling pathway in ALI after the ‘secondhit’, we examined the lung tissue histologically. Histological examinations showedremarkable lung injuries in the tissues harvested8h after MDP injection, while theinjuries stayed similar at24h. In contrast, when mice were pretreated with3-MA toblock autophagy, WT lung injuries aggravated with time during24h after the ‘secondhit’ while the NOD2-/-lung injuries stayed similar. These data indicated that NOD2played a key role in mediating lung injury and autophagy possessed ananti-inflammatory function. To study the effect of NOD2-autophagy signalingpathway on the myeloperoxidas (MPO) level in lung tissue from mice undergoing‘second hit’, we used the HS/R+MDP model and examined the activity of MPO in thelung tissue. The results showed that lung injury caused by neutrophils wasexacerbated when autophagy was blocked. Defect in NOD2expression could blockthe downstream inflammatory signals and thus relieve the lung inflammation causedby ‘second hit’. To investigate how the NOD2-autophaty signaling pathwaymodulates the inflammatory cytokines in lungs from ‘second hit’ model, we measuredIL-6and IL-12in the BAL fluid. Results showed that lung inflammation aggravatedwhen autophagy was blocked. To confirm effect of NOD2-autophagy of AM on theexpression and release of inflammatory cytokines, we performed the ‘second hit’model and measured the mRNA level of TNF-α, IL-6and IL-12in AM.Part5. Mechanisms of NOD2-autophagy modulation of ALI after‘second hit’To study NOD2-autophagy modulation on AM apoptosis after ‘second hit’ with MDPand its role in ALI after ‘second hit’, we cultured WT and NOD2-/-AM and treatedwith HMGB1and MDP with/without3-MA. We found with TUNNEL staining that 24h after treatment, WT cells underwent apoptosis when treated with MDP alone orHMGB1+MDP, with HMGB1+MDP resulting in profound apoptosis. When treatedwith3-MA, apoptosis decreased significantly. NOD2-/-AM showed little signs ofapoptosis. To test these findings in vivo, we used HS/R+MDP ‘second hit’ modelwith/without3-MA administration, in WT or NOD2-/-mice. We found24h after‘second hit’ WT AM underwent a significantly high level of apoptosis (18.8±1.1%)comparing with cells from mice treated with3-MA (3.3±0.4%). AM from NOD2-/-mice underwent relatively low levels of apoptosis,2.0±0.1%or0.5±0.2%when3-MA was injected or not, respectively.SummaryBased on the findings above, we conclude:1, HS/R upregulate NOD2expression inAM through HMGB1-TLR4signaling pathway;2, HS/R upregulated NOD2expression in AM and thus promote its sensitivity to MDP and therefore aggravate thelung inflammation and lung injury in the HS/R+MDP ‘second hit’ model;3, HS/Rinduces lung inflammation and lung injury in an IL-1β dependent manner, which ismodulated by NOD2;4, HS/R-mediated NOD2upregulation induces autophagy inlung inflammation and lung injury;5, NOD2-autophagy signaling pathway mediateslung inflammation and lung injury in the HS/R+MDP ‘second hit’ model;6,NOD2-autophagy signaling pathway plays Janus role in the HS/R+MDP ‘second hit’model, being both proinflammatory and anti-inflammatory;7, NOD2-autophagy maysuppress ‘second hit’-induced lung inflammation by promoting AM apoptosis.In brief, this study provides a novel mechanism by which ‘second hit’ induces ALI:NOD2-autophagy signaling pathway initiates and modulates ‘second hit’-induced ALI,which may provide a novel target for clinical intervention.