In this study, we present the effect of IL-1 on primary cilia length and highlight, for the first time, the potentially fundamental role of IFT-mediated primary cilia elongation in the progression of inflammation. Further to this, we show that some of the molecular mechanisms highlighted in previous studies apply to this cytokine-induced lengthening. Most interestingly of all, we demonstrate the key role for the cilium and IFT in downstream inflammatory signaling.
By their quiescent nature, freshly isolated primary chondrocytes represent an excellent and highly relevant non-proliferative primary cell model for studying ciliogenesis in the context of inflammation. Freshly isolated chondrocytes expressed primary cilia with a mean length of ~2 μm in monolayer cultures, (Fig. a). For comparison, in healthy bovine patellae cartilage, mean lengths of 1.1–1.5 μM, are observed in the superficial to deep zones, respectively [26
]. In situ measurements of cilia length within cartilage tissue are difficult given the three dimensional orientation of the cilia and the resolution of confocal microscopy in the z
axis. By contrast, the use of isolated chondrocytes cultured in monolayer provides a simple, accurate, and reproducible measurement of primary cilia length.
Our results indicate that the pro-inflammatory cytokine, interleukin-1, stimulates cilia elongation. It is most likely that IL-1 is having an effect on pre-existing cilia rather than holding cells in G0 for longer. Cilia length has been shown to be regulated by a host of genes, proteins, and signaling cascades [13
], but never by exposure to inflammatory cytokine. Cilia elongation occurred after just 3 h of IL-1 exposure and at the lowest concentration tested (2 ng.mL−1
). Both IL-1β and the cell-associated form IL-1α stimulate elongation. In other tissues, fibroblasts are thought to be intermediaries in the immune reaction during an inflammatory progression that includes chemokine signaling via prostaglandins and nitric oxide release [41
]. We show that this phenomenon of cilia elongation, in response to the inflammatory cytokine IL-1, is not just active in chondrocytes but is also present in fibroblasts and therefore may have implications for all inflamed tissues.
Previous pharmacological work in multiple cell types [14
] has indicated a role for adenylate cyclase-cAMP and PKA in the extension of cilia. In synovial fibroblasts, the inhibition of adenylate cyclase, by lithium, was shown to elongate cilia [35
]. Moreover, in these studies, the activity of adenylate cyclase interfered with the effects of lithium on cilia length. Conversely, however, in kidney and bone cells, the activation of adenylate cyclase by forskolin resulted in cilia elongation [14
]. Our results indicate firmly that PKC, MEK–ERK, and PKA are involved in IL-1-induced length increases (Fig. ), but the role for cAMP was not found (Fig. ). We found that both introduction of an analogue of cAMP and inhibition of adenylate cyclase induced cilia elongation both in isolation and supplementary to the effects of IL-1. We found inhibition of upstream Gαi subunits also elongated cilia in isolation, but these effects did not supplement those of IL-1. This may indicate a shared role of G-protein subunits in IL-1 and adenylate cyclase mechanisms. However, importantly, we see no evidence to suggest that IL-1-induced elongation and cAMP-induced elongation are linked upstream of PKA (Fig. ). The complex roles of adenylate cyclase isoforms localized to the cilium [35
] and localized changes in cAMP may explain the apparent conflicts from previous studies. Adenylate cyclase isoforms are exhibited by chondrocytes [42
], and we have shown that the system is active in chondrocytes where a cAMP analogue elongates the cilium in a dose-dependent manner acting through PKA, but have no evidence for a relevance of this in the context of Inflammation.
Fig. 6 Schematic summary of the proposed pathways behind IL-1 influence on chemokine release via ciliary elongation, as indicated by pharmacological and genetic experiments. IL-1 exerts positive influence on cilia elongation via PKA, MEK–ERK, and PKC. (more ...)
We thus hypothesize that, in response to IL-1, downstream regulation of anterograde IFT is conducted by PKA, as has been shown previously with fluid flow mediated changes in cilia length [14
]. Indeed, the cilia role of PKA is well established, as PKA has been linked to cilia-dependent signaling systems including hedgehog and polycystin signaling [43
]. In addition to the role of PKA, we have established that PKC and the MEK–ERK kinases are both involved in IL-1-stimulated elongation, perhaps a further indication of the complexity of IL-1-elicited cellular responses. Other potential candidates involved in IFT and IL-1-induced cilia elongation include hypoxia inducible factor (HIF), [28
], cytosolic calcium, and the actin cytoskeleton [14
], all of which are influenced by interleukins [49
]. However, this is not to exclude additional reported mechanisms including FGF signaling [50
], the Dcdc2 protein [13
], and the tubulin cytoskeleton itself [23
]. We believe that the significance of IL-1 regulation of ciliary length may lie with hedgehog signaling alterations as supported by the correlation between structural changes, including length, and hedgehog signal transduction [36
To investigate the role of IFT and primary cilia in downstream inflammatory signaling, the present study utilized an IFT88 mutant chondrocyte model. The Tg737ORPK
model knocks out the IFT88 gene, disrupting polaris expression, and rendering cells with stunted [10
] or, in the case of chondrocytes here, no obvious cilia structure (Fig. b). In response to IL-1, many cells including chondrocytes exhibit induction of nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX-2) expression triggering the release of the potent inflammatory chemokines, nitric oxide (NO) and prostaglandin (PGE2
). Here, we show that loss of primary cilia and IFT88 had no effect on basal NO and PGE2
, but significantly attenuated the normal up-regulation observed in WT cells in response to IL-1β (Fig. ). This may explain why mechanical loading, which is known to reduce cilia length in chondrocyte [30
], also down-regulates the IL-1β-induced release of NO and PGE2
]. It is also likely that IL-1β-induced cilia elongation will influence other aspects of cilia function including mechanotransduction [9
] and hedgehog signaling which has already been linked to arthritis [27
]. We finally examined the link between IL-1-induced cilia elongation via PKA (Fig. a) and the role of IFT88 (Fig. c, d) in the inflammatory response (Fig. e, f). We show that selective inhibition of PKA also inhibits PGE2
and NO release in bovine chondrocytes treated with IL-1. Thus, the cilium and associated elongation in response to IL-1 play critical roles in the activation of inflammatory chemokine signaling. The primary cilium already has central roles throughout cell biology, but here we propose, for the first time, that the cilium and the regulation of its structure and function are of fundamental importance in inflammation. The localized and specific nature of the cilia proteome governing maintenance and function of the primary cilia provides exciting potential therapeutic targets for inflammatory conditions.