Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial inflammation and progressive destruction of the articular tissue. It is well known that continuous and intense nociceptive input from the inflamed joint may induce the hyperexcitability of spinal dorsal horn neurons (a process known as central sensitization) and hyperalgesia (an enhanced response to noxious stimuli) ([1-3]). Intriguingly, the increased excitation of the spinal cord can also signal back to the periphery via a variety of neuronal pathways, such as the autonomic nervous system (), dorsal root reflexes ([5, 6]), and hypothalamic–pituitary–adrenal (HPA) axis, to control peripheral inflammation (). There is convincing evidence to support the idea that a variety of spinally administered compounds attenuate peripheral inflammation. These compounds include adenosine (), serotonin (), ketamine and morphine (), thalidomide (), antagonists of microglia and inhibitors of astrocytes (), and blockers of p38 MAPK ().
NF-κB is a transcription factor that plays a pivotal role in the central nervous system (CNS), in processes such as inflammation, neuronal plasticity, synaptic transmission, learning, memory, and pain (). NF-κB contributes to the regulation of these CNS processes by positively regulating the transcription of numerous genes, including cytokines (interleukin-1β [IL-1β], IL-6, and tumor necrosis factor α [TNFα]), proinflammatory enzymes (cyclooxygenase 2 [COX-2] and inducible nitric oxide synthase [iNOS]), chemokines, and adhesion factors (). Five subunits of NF-κB have been identified, namely, gp105/p50 (NF-κB1), p100/p52 (NF-κB2), p65 (RelA), RelB, and c-Rel (). The active form of NF-κB is a dimer formed from 2 of these subunits. The most common and best-characterized form of NF-κB is the p50/p65 heterodimer, which is widely expressed in the CNS and plays an important role in the regulation of gene expression ().
Previous studies have indicated that activation of spinal NF-κB/p65 occurs in peripheral tissue damage or inflammation ([17, 18]). Findings in a recent study from our group showed that intrathecal injection of a lentiviral vector, LV-shNF-κB/p65, an inhibitor of NF-κB/ p65 that encodes short hairpin RNAs (shRNAs) targeting NF-κB/p65, significantly reduced the expression of spinal NF-κB/p65 and also reduced mechanical and thermal hyperalgesia following peripheral nerve injury (). These findings suggest that NF-κB/p65 has a role in central sensitization. However, it is still unknown whether spinal NF-κB/p65 can also facilitate a peripheral inflammatory response.
Therefore, the goal of the present study was to test whether the inhibition of spinal NF-κB/p65 expression could significantly influence the progression and severity of peripheral inflammation and hyperalgesia, using an experimental arthritis model of rat adjuvant-induced arthritis (AIA). We also evaluated the expression of IL-1β, TNFα, and COX-2 in the spinal cord of these rats to gain insight into the mechanisms involved in the contributions of NF-κB/p65 to peripheral inflammation and hyperalgesia in this model system.
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The aim of the current study was to evaluate the role of spinal NF-κB/p65 in the regulation of peripheral inflammation and hyperalgesia resulting from induction of arthritis in rats. To this end, we knocked down the expression of NF-κB/p65 using LV-shNF-κB/p65, which was administered intrathecally into lumbar segments of the spinal cords of rats. We observed that intrathecal pretreatment with LV-shNF-κB/p65 on day 3 following CFA injection caused a significant reduction in the hyperalgesia, paw edema, and joint destruction in rats with AIA. This indicates that spinal NF-κB/p65 has a pivotal role in the generation of peripheral inflammation and hyperalgesia. Moreover, we also found that intrathecal delivery of LV-shNF-κB/p65 10 days post–CFA injection was able to partially reverse the already-established hyperalgesia and paw edema. This indicates that NF-κB/p65 activation was still ongoing, and that inhibiting the NF-κB/p65 pathway could disrupt the development of painful inflammatory disorders.
Peripheral tissue damage or inflammation induces a series of activation events in the spinal cord. During the development of experimental arthritis, the peripheral nociceptors may be sensitized by inflamed synovium and damaged articular tissue ([27, 28]), and continuous and intense nociceptive input from inflamed joints may induce neural–immune interactions ([12, 29]). This leads to the production and secretion of cytokines, excitatory amino acids, COX-2, and prostaglandins, which can increase the excitability of nociceptive neurons at the spinal level, including the central terminals of the primary sensory afferents (i.e., central sensitization) ([30, 31]). In addition, the spinal cord can also signal to the periphery to regulate inflammation.
A variety of neuronal pathways that modulate peripheral inflammation have been implicated, including the sympathetic and the parasympathetic branches of the autonomic system (). The branches of the vagal nerve and sympathetic fibers innervate immune organs, wherein they can influence peripheral immune responses. Another mechanism, the dorsal root reflex, involves antidromic signaling along somatic afferent fibers that influence peripheral inflammation by releasing neuropeptides, such as vasoactive intestinal peptide, substance P, and calcitonin gene–related peptide, from the sensory nerve endings ([5, 6]). Moreover, the adrenal cortex (the HPA axis), which can also provide a feedback mechanism in the processes of inflammation, is often blunted in a wide range of autoimmune and inflammatory diseases such as rheumatoid arthritis (). Previous studies have supported this notion. For instance, spinal cord MAPK and specific cytokines such as IL-1β and TNFα, which can be regulated by NF-κB, are involved in the regulation of peripheral inflammation ([13, 30, 32]).
The NF-κB family of transcription factors controls the expression of genes that are critical for inflammation and immune activation (). Family members of NF-κB in the articular tissue can play a vital role in the initiation and development of arthritis by the regulation of cytokines, such as IL-1β, IL-6, and TNFα, and the regulation of enzymes involved in tissue remodeling, such as COX-2, iNOS, and matrix metalloproteinase 9 ([34, 35]). Interestingly, NF-κB is also activated in the spinal cord in response to peripheral nerve injury or tissue inflammation. In the rat chronic constriction injury (CCI) model, we previously observed extensive colocalization of NF-κB/p65 with TNFα in the spinal dorsal horn (), and down-regulation of spinal NF-κB/p65 expression significantly attenuated sciatic nerve ligation–induced mechanical and thermal hyperalgesia (). In addition, spinal NF-κB is also activated following injection of CFA or formalin into the footpad, and spinal application of an NF-κB inhibitor reduces pain-related behavior ([18, 37, 38]). Similar to the findings in these studies, the present results show that peripheral inflammation resulting from AIA led to the activation of NF-κB/p65 in the spinal cord, and a spinally delivered inhibitor of NF-κB/p65 expression, the lentiviral vector LV-shNF-κB/p65, significantly attenuated the inflammation-induced hyperalgesia in this rat model of arthritis.
Since spinal NF-κB/p65 has been shown to be activated following peripheral nerve tissue injury or inflammation, and since it is involved in the induction of central sensitization and hyperalgesia, we explored the possibility that spinal NF-κB/p65 regulates peripheral inflammation. In the present study, the spinally delivered inhibitor LV-shNF-κB/p65 markedly decreased the severity of paw edema, inflammatory cell infiltration, and destruction of cartilage and joint structures in rats with AIA. In contrast, spinal delivery of LV-NC had no effect on the paws of arthritic rats. In addition, our findings indicated that NF-κB/p65 was expressed in neurons and astrocytes in the spinal dorsal horn, as observed on day 14 after CFA injection.
A recent study demonstrated that the up-regulation of spinal NF-κB in rats with CCI could be abated by systemic treatment with MK-801 (), an N-methyl-d-aspartate (NMDA) receptor antagonist known to improve central sensitization and hyperalgesia in rats (). NMDA receptor activation may activate NF-κB in neurons ([39, 41, 42]), suggesting that there is some involvement of neuronal NF-κB in central sensitization. On the other hand, previous studies have shown that selective inhibition of NF-κB activity in astrocytes in the spinal cord significantly reduced the extent of hyperalgesia and allodynia after formalin injection (), which suggests a role for NF-κB in the modulation of astrocytes following peripheral inflammation. Therefore, NF-κB up-regulation in the spinal cord after peripheral nerve injury or inflammation may reflect changes in both neuron and astrocyte function.
Moreover, we found that the expression levels of IL-1β, TNFα, and COX-2 protein were highly up-regulated in the spinal cord of rats with AIA. In the family of proinflammatory cytokines, TNFα and IL-1β have long been strongly implicated in the modulation of peripheral pain and inflammation of the joints. In addition to their peripheral action, TNFα and IL-1β are up-regulated in the spinal cord following peripheral inflammation, and both are known to be involved in the development and maintenance of experimental arthritis ([11, 13, 30, 32]). COX-2, which is constitutively expressed in the spinal cord, is a major contributor to the induction of spinal prostaglandin E2 (). It has been shown that peripheral inflammation can up-regulate the expression of COX-2 in the spinal cord (), and spinal administration of the COX inhibitor indomethacin attenuated inflammatory edema (). Our results demonstrate that spinally delivered LV-shNF-κB/p65 markedly reduced the overexpression of spinal TNFα, IL-1β, and COX-2 in rats with AIA. These findings are consistent with those reported previously ([19, 37]).
Interestingly, in addition to being regulated by NF-κB, spinal TNFα, IL-1β, and COX-2 can help to propagate the extension of the neuroinflammatory response by activating NF-κB ([47-49]), which forms a positive feedback mechanism to exaggerate the inflammatory process. Thus, NF-κB activation may amplify/perpetuate the neuroinflammatory responses, which may act directly on the central terminals of primary afferent neurons and on dorsal horn neurons and thereby contribute to central sensitization and hyperalgesia. We therefore propose that the analgesic and antiinflammatory effects of LV-shNF-κB/p65 might be mediated, at least in part, through the prevention of neuronal and astrocytic NF-κB/p65 and subsequent suppression of the positive feed-forward loop between NF-κB/p65 and inflammatory mediators such as TNFα, IL-1β, and COX-2.
In conclusion, our findings indicate a pivotal role for spinal NF-κB/p65 in the initiation and development of both peripheral inflammation and inflammation-related hyperalgesia. Reduction in the expression of NF-κB/p65 protein in the spinal neurons and astrocytes markedly reduced both phenomena. Thus, interference with NF-κB/p65 on the spinal level may provide a novel treatment option for painful inflammatory disorders.