Cell autonomous expression of inflammatory genes in biologically aged fibroblasts associated with elevated NF-kappaB activity

Chronic inflammation associated with the aging process has been implicated in a host of degenerative disease states including osteoarthritis, atherosclerosis, type-2 diabetes and even cancer [1–3]. Age-associated chronic inflammatory states are distinct from inflammation triggered by infection. It is presently unclear to what extent chronic inflammatory states in older individuals represent autoimmune processes caused by deregulation of the immune system [4, 5]. Alternatively, these states may arise as a consequence of an increased cell stress response in old cells triggered by molecular damage incurred over a lifetime. In support of cell autonomous causes for age-associated inflammation, expression of inflammatory markers, such as cytokines, has been observed in cells subjected to replicative senescence in vitro caused by serial passaging [6–9]. However, molecular events observed during replicative senescence in vitro do not necessarily mirror events that occur in human aging, which is of a dramatically different time frame. This consideration motivated the present investigation of age-associated changes in proliferating fibroblasts derived from donors at different biological ages. Only few reports using fibroblasts aged in vivo have been published and these reports largely focused on age-associated changes in cell cycle progression of dividing cells [10, 11].

In contrast, in the present study we focused on 'inflammatory signatures', i.e. changes in gene expression patterns previously implicated in inflammatory states. Furthermore, we considered that determination of cell states associated with the aging process should be performed in quiescence rather than exponentially growing fibroblast cultures. This was based on the consideration that, under physiological conditions in tissues in vivo, the majority of fibroblasts neither proliferate nor have achieved replicative senescence akin to that of cultured fibroblasts. Therefore, we investigated differences in gene expression profiles of primary human fibroblasts derived from donors at different biological ages and rendered quiescent by growth factor starvation [12]. We report that expression of mRNA transcripts encoding proteins with roles in inflammation is elevated in fibroblasts derived from older individuals. This gene expression signature is associated with increased DNA binding of transcription factor nuclear factor kappa B (NF-κB) in a subset of aged cells and plays a role in mediating inflammatory responses.

Activation of the latent transcription factor NF-κB plays a key role in mediating inflammatory responses and gene expression patterns [21]. It has also been demonstrated that genetically blocking NF-κB in the skin of aged mice reverses the gene expression pattern seen in aged cells as well as reverts the appearance of tissues to that of younger skin samples, illustrating the critical role of NF-κB in the aging phenotype [22, 23]. Here we show that in human aging NF-κB becomes constitutively active, mediating enhanced transcription of inflammatory markers. Cytokines, chemokines, and components of the complement cascade dominated the upregulated genes. This is the first report to demonstrate that a low-grade inflammatory state, as it relates to aging, occurs in cell lines irrespective of the presence of other cell types or the in vivo context, which highlights that this inflammatory state is intrinsic and cell autonomous. While NF-κB appears to be an important regulator, the profile of inflammatory genes expressing at higher levels is still specific for each sample investigated and not all genes are regulated by NF-κB, suggesting that additional factors may play a role mediating the profile.

It was reported that protein abundance of inflammatory markers parallels gene expression [24]; therefore it can be assumed that most inflammatory markers shown here would indeed contribute to the activation of the immune system, giving rise to a chronic inflammatory state in vivo. Specifically, fibroblasts acting as 'immune-competent' cells show higher levels of activity of molecules involved in antigen recognition, presentation and destruction such as FGR3B, DF and C1R. Essentially, aging cells send a destructive 'non-self' message to the immune system, for which rheumatoid arthritis is a prime example. The production of cytokines may establish a positive feedback loop since these markers can also activate NF-κB, and herewith increase and prolong chronic inflammation [25]. Similarly, interferons, known to be produced by synovial fibroblasts, have been associated with prolonged T cell survival in rheumatic joints [26].

Multiple factors can contribute to the activation and post-translational modification of NF-κB [21, 27], but as it relates to aging, cell-intrinsic factors can be traced back to mitochondrial dysfunction. It has been recognized that mitochondrial deficiency accompanies the aging process, albeit the exact mechanisms for mitochondrial dysfunction discussed, for example mtDNA mutations in vertebrates, remain controversial [28–31]. Since we did not find indications for increased levels of reactive oxygen species (ROS) and related expression of scavenger molecules (data not shown), we hypothesize that activating mechanisms of NF-κB, as a consequence of mitochondrial dysfunction, may include other mechanisms such as accumulation of oxidized proteins and lipids in aged fibroblasts [32] or disturbances of intracellular calcium homeostasis [33, 34]. Further, intracellular NADH abundance that has been related to defects in mitochondrial respiration is accompanied by inactivation of phosphatase and tensin homolg gene (PTEN). PTEN is a negative regulator of the Akt pathway [35, 36], while Akt is known to converge with NF-κB signaling [35]. Insofar, NF-κB may participate in a retrograde response and cross-talk between dysfunctional mitochondria and nuclear genes as a cellular pro-survival mechanism. This would also explain why anti-inflammatory treatment regimes lead only to a temporary relief of symptoms, but would not correct for the primary cause of inflammation, which has its roots in cellular aging.

This study is a cross-sectional, rather a longitudinal study and the definition of age-groups follows the available samples in fulfillment of the stringent criteria set forth in the Method section. Variability as seen here is rooted in the process of biological aging, which is different from chronological age, and in cell selectivity during the initial establishment of cell cultures from biopsies. We also recognize that a study in vitro cannot capture the more complex interactions in vivo, involving other cellular participants and components of the immune system, which provoke, mediate, and amplify the primary cellular response. However, chronic inflammation has been implicated in many aging-associated conditions [1, 37], and inflammatory markers described here have been reported in other age-related studies in which gene expression analysis was performed such as in brain [38], lung [39], liver [40], kidney [41] and coronary arteries [42]. Moreover, consensual findings show upregulation of NF-κB activity as a pivotal mediator of aging related inflammation in rodent and human tissues [22], including mouse brain, muscle [43–46] and human endothelial tissues [47], which suggests that similar processes as those observed here play a role in many other cell types and tissues.

Overall this study provides evidence for a cell-intrinsic activation of NF-κB and related upregulation of inflammatory markers. While inflammation is a protective host response against harmful external stimuli, in the context of biological aging this once beneficial response becomes constitutively active, potentially as a consequence of mitochondrial dysfunction. This may in turn mediate susceptibility to age-related diseases in tissues since aberrant expression of inflammatory markers plays a key role in pathogenesis and tumorgenesis. Our study may provide a preliminary diagnostic tool to profile aging at the cellular level.