Transcription factor IRF5 drives P2X4R+-reactive microglia gating neuropathic pain

Masuda, Takahiro; Iwamoto, Shosuke; Yoshinaga, Ryohei; Tozaki-Saitoh, Hidetoshi; Nishiyama, Akira; Mak, Tak W.; Tamura, Tomohiko; Tsuda, Makoto; Inoue, Kazuhide

doi:10.1038/ncomms4771

Download PDF

Article
Open access
Published: 13 May 2014

Transcription factor IRF5 drives P2X4R⁺-reactive microglia gating neuropathic pain

Takahiro Masuda^1,2,
Shosuke Iwamoto¹,
Ryohei Yoshinaga¹,
Hidetoshi Tozaki-Saitoh^1,2,
Akira Nishiyama³,
Tak W. Mak⁴,
Tomohiko Tamura³,
Makoto Tsuda¹ &
…
Kazuhide Inoue^1,2

Nature Communications volume 5, Article number: 3771 (2014) Cite this article

8983 Accesses
137 Citations
4 Altmetric
Metrics details

Subjects

Abstract

In response to neuronal injury or disease, microglia adopt distinct reactive phenotypes via the expression of different sets of genes. Spinal microglia expressing the purinergic P2X4 receptor (P2X4R) after peripheral nerve injury (PNI) are implicated in neuropathic pain. Here we show that interferon regulatory factor-5 (IRF5), which is induced in spinal microglia after PNI, is responsible for direct transcriptional control of P2X4R. Upon stimulation of microglia by fibronectin, IRF5 induced de novo expression of P2X4R by directly binding to the promoter region of the P2rx4 gene. Mice lacking Irf5 did not upregulate spinal P2X4R after PNI, and also exhibited substantial resistance to pain hypersensitivity. Furthermore, we found that expression of IRF5 in microglia is regulated by IRF8. Thus, an IRF8-IRF5 transcriptional axis may contribute to shifting spinal microglia toward a P2X4R-expressing reactive state after PNI. These results may provide a new target for treating neuropathic pain.

Galectin-3 activates spinal microglia to induce inflammatory nociception in wild type but not in mice modelling Alzheimer’s disease

Article Open access 22 June 2023

George Sideris-Lampretsas, Silvia Oggero, … Marzia Malcangio

Sigma-1 receptor expression in a subpopulation of lumbar spinal cord microglia in response to peripheral nerve injury

Article Open access 07 September 2023

Ethan Schonfeld, Thomas Michael Johnstone, … Anand Veeravagu

Microglia and macrophages promote corralling, wound compaction and recovery after spinal cord injury via Plexin-B2

Article 28 February 2020

Xiang Zhou, Shalaka Wahane, … Hongyan Zou

Introduction

Microglia are the resident immune cells of the central nervous system (CNS). Under physiological conditions, microglia actively move their branched processes to sense pathological alterations or disturbances to ultimately maintain CNS homeostasis^1,2,3. Neuronal damage transforms microglia into reactive phenotypes with biochemical alterations, such as the activation of gene transcription^1,2,4,5. These alterations have been implicated in the disruption of the CNS environment, which is linked to the pathogenesis of various CNS diseases^1,4,5. The state of microglial activation is regulated by extracellular signals on surface receptors and also tightly controlled by transcription factors^6,7. We have previously identified interferon regulatory factor-8 (IRF8) as a crucial transcription factor that activates the expression of a variety of genes associated with the activated processes of microglia⁸. However, little is known about the transcriptional regulatory mechanisms underlying the phenotype shift towards a specialized reactive state of microglia.

Injury to the nervous system as a consequence of multiple sclerosis, diabetes mellitus, cancer or traumatic injury often causes a debilitating chronic pain syndrome, termed neuropathic pain^9,10,11, which is refractory to currently available treatments¹¹. Accumulating evidence indicates the pivotal roles of spinal microglia in neuropathic pain. Following peripheral nerve injury (PNI), microglia in the spinal dorsal horn exhibit an reactive phenotype and upregulated expression of a variety of genes, including purinergic P2X4 receptor (P2X4R)^8,12,13. Stimulating these receptors on microglia causes the release of brain-derived neurotrophic factor (BDNF)^14,15, which in turn disinhibits the inhibitory system of the dorsal horn nociceptive network¹⁴ or enhances excitatory synaptic transmission in the dorsal horn neurons of the spinal cord¹⁶. The pathological alterations convert innocuous inputs to a nociceptive signal output to the brain, thus contributing to pain hypersensitivity¹³. Therefore, a specific phenotype of spinal microglia characterized by high expression of P2X4R (P2X4R⁺ microglia) after PNI is crucial for the pathogenesis of neuropathic pain^10,13,15. However, the molecular machinery that underlies the formation of P2X4R⁺ microglia remains poorly understood.

Here we identify an interferon regulatory factor-5 (IRF5) as a microglial gene whose expression is induced by PNI. IRF5 induces de novo expression of P2X4R in activated microglia by fibronectin through a direct binding to the promoter region of the P2rx4 gene. IRF5-deficient mice show no upregulation of spinal P2X4R after PNI and substantial resistance to pain hypersensitivity. IRF5 is directly regulated by IRF8 in microglia. Thus, our findings indicate that an IRF8–IRF5 transcriptional axis is a critical regulator for shifting towards a P2X4R⁺-reactive phenotype that gates neuropathic pain after PNI and may provide a new target for treating neuropathic pain.

Results

PNI induces expression of IRF5 in spinal microglia

To uncover transcription factors that control reactive states of microglia, we initially performed a genome-wide microarray analysis from the spinal cord of wild-type (WT) mice with PNI, which exhibits injured nerve projections and activated microglia, and sorted out the transcription factors upregulated. Among genes evaluated in three or four independent analyses, we identified IRF5 (P<0.001) and IRF9 (P=0.016) as significant transcription factors increased in the spinal cord ipsilateral to nerve injury (Supplementary Fig. 1a). IRF5 showed a more significant rate of expressional change with high reproducibility compared with that of IRF9 (Supplementary Fig. 1a); we therefore focused on IRF5 in the present study. In the periphery, IRF5 plays pivotal roles in the immune system, such as the induction of pro-inflammatory cytokines¹⁷ and polarization of M1 macrophages¹⁸. However, the nature of IRF5 in the CNS is entirely unknown.

To determine the cell types expressing IRF5 in the spinal cord after PNI, we examined the localization of Irf5 mRNA using in situ hybridization combined with immunofluorescence of the cell-type-specific markers. On sections of the fourth lumbar (L4) spinal cord of mice, few or no detectable signals of Irf5 mRNA were found on the contralateral side. However, PNI increased the signal intensity of Irf5, with punctate distribution in the ipsilateral dorsal horn (Fig. 1a, intense violet dots indicated by arrowheads). These signals, which were not observed when hybridized with a corresponding sense probe (Supplementary Fig. 1b), were highly restricted to cells labelled with ionized calcium-binding adapter molecule-1 (Iba1; microglial marker), but not with glial fibrillary acidic protein (GFAP; astrocytic marker) (Fig. 1b). Most of Iba1-positive microglia showed the signals of Irf5 mRNA in the ipsilateral dorsal horn (42/49 Iba1-positive cells). These results suggest that in the spinal cord, Irf5 expression is selectively induced in microglia after PNI.

**Figure 1: PNI induces the expression of IRF5 in spinal microglia.**

We then determined the temporal expression pattern of Irf5 mRNA in the spinal cord following PNI via real-time PCR analysis. In support of the in situ hybridization results, expression levels of Irf5 mRNA were low in the spinal cord of naive (that is, before PNI) mice, but markedly increased in the spinal cord ipsilateral to the injury (Fig. 1c). This increase occurred from postoperative day 1, and plateaued by day 3 for >2 weeks (Fig. 1c). In contrast, no detectable change was found in the contralateral spinal cord (Fig. 1c). Western immunoblot analysis using a specific antibody for IRF5 (Supplementary Figs 1c and 13), showed an upregulation of IRF5 protein in the spinal cord after PNI, with a time course and a bilateral difference matching those of Irf5 mRNA (Fig. 1d,e). Overall, these results indicate that IRF5 is highly restricted to microglia of the dorsal horn, and is dramatically increased after PNI, persisting in the cell for at least 2 weeks.

Fibronectin-mediated upregulation of P2X4R requires IRF5

To explore the function of IRF5 in microglia, we first transduced microglial BV2 cells with a lentiviral vector encoding IRF5, followed by the assessment of the levels of gene transcripts. However, ectopic expression of IRF5 resulted in the minimal effect on an expressional pattern of microglial genes (Supplementary Fig. 2), which is consistent with previous reports noting that full-length IRF5 by itself has a lower potency to drive the reporter gene^19,20. IRF5 is known to predominantly reside in the cytoplasm, and during stimulation-dependent activation, it translocates to the nucleus to activate gene transcription¹⁹. Thus, to reveal an inherent mechanism that activates microglial IRF5, we focused on several signalling mediators that have been implicated in microglial activation, including toll-like receptor-4 (TLR4)²¹, interferon-gamma (IFN-γ)²², adenosine triphosphate (ATP)^23,24 and fibronectin^25,26. These mediators were screened by evaluating their abilities to induce translocation of IRF5. Early reports have shown that IRF5 in macrophages is activated by stimulating TLRs^17,20. In the present study, however, the TLR4 ligand lipopolysaccharide (LPS) unexpectedly did not change the localization of IRF5 in microglia (Supplementary Figs 3 and 14). Likewise, neither ATP nor IFN-γ induced IRF5 translocation to the nucleus (Supplementary Figs 3 and 14). In contrast, IRF5 protein accumulated in the nuclear fraction when cells were stimulated with fibronectin (Fig. 2a,b), and this effect inversely correlated with the reduced level of cytosolic IRF5 (Fig. 2a,b). Total IRF5 protein in whole-cell lysate of microglial cells did not differ before and after treatment with fibronectin (Fig. 2a,b). Therefore, these results indicate that fibronectin activates IRF5 in microglia.

**Figure 2: IRF5 is required for fibronectin-mediated upregulation of P2X4R in microglia.**

Fibronectin is increased in the spinal cord after PNI²⁵ presumably through release from astrocytes²⁷ or extravasation from blood flow²⁸, and plays crucial roles in de novo expression of P2X4R in microglia^25,26,29. Therefore, IRF5 may regulate P2X4R expression in microglia. To test this hypothesis, we used BV2 cells in which IRF5 expression had been knocked down by short hairpin RNA (shRNA) targeting IRF5 (Supplementary Fig. 4a,c). We found that IRF5 shRNA markedly suppressed fibronectin-induced upregulation of P2X4R at both the mRNA and protein levels (Fig. 2c–e) without any alteration in the basal expression of P2X4R (Supplementary Fig. 4b), suggesting IRF5-mediated expression of P2X4R in microglia. To determine whether IRF5 directly regulates the transcription of P2X4R, we examined the recruitment of IRF5 to the promoter loci of P2X4R by chromatin immunoprecipitation (ChIP)-qPCR analysis. Using the online software, Genomatix MatInspector ( www.genomatix.de/index.html), we identified four regions that contained an IRF-binding motif, namely interferon-stimulated response element (ISRE), in the promoter of mouse P2rx4 gene (Fig. 2f). A ChIP-qPCR assay revealed that a fragment of the P2rx4 promoter containing each putative binding site was amplified with the complex immunoprecipitated with the IRF5 antibody (Fig. 2g). Importantly, the degree of amplification was enhanced by fibronectin treatment (Fig. 2g), implying that activated IRF5 binds to these p2rx4 promoter regions. In contrast, the amplification rates of each complex immunoprecipitated with antibodies for the other IRF members were not changed by fibronectin treatment (Fig. 2g). Collectively, these findings suggest that IRF5 functions as a transcriptional activator on the P2rx4 promoter leading to de novo expression of P2X4R in microglia (that is, IRF5 shifted microglia towards the P2X4R⁺ phenotype).

IRF8 directly regulates IRF5 in microglia

We have previously shown that IRF8 in mice spinal cord is markedly upregulated in microglia, and plays a crucial role in their activation⁸. Our results showed that the expression pattern of IRF8 in the spinal cord resembled that of IRF5 (Fig. 1). We therefore examined the hierarchy and interaction between IRF5 and IRF8 in microglia. BV2 cells transduced with green fluorescent protein (GFP)-tagged IRF8 (IRF8-GFP)⁸ significantly increased the expression of IRF5 in a manner that was dependent on the level of expression of IRF8 (Fig. 3a,c). However, the mutant IRF8 in which lysine at amino acid position 79 was replaced with Glu (IRF8(K79E)), resulting in a lack of DNA binding activity³⁰, failed to increase IRF5 (Fig. 3b,d). IRF8-mediated upregulation of IRF5 expression was verified using primary cultured microglia transduced with IRF8-GFP (Fig. 3e). We further investigated whether IRF8 directly binds to the promoter loci of IRF5. Previous experiments by genome-wide ChIP coupled to DNA sequencing (ChIP-seq) assay in myeloid progenitor cells revealed two salient IRF8 ChIP-seq peaks localized on the promoter region of IRF5 (ref. 31). We then examined whether IRF8 binds to these sites (regions 1 and 2; Fig. 3f) in microglial cells by ChIP-qPCR analysis, and validated binding of IRF8 to predicted binding sites for IRF5 (Fig. 3g). These binding intensities were much higher in cells transduced with IRF8-GFP compared with normal cells (Fig. 3g). In contrast, binding activities of the other IRF members were indistinguishable between the two types of cells (Fig. 3g). To confirm whether IRF8 transactivates Irf5 gene expression through these binding sites, we performed a luciferase assay using a reporter containing the two target regions of the mouse Irf5 gene (Fig. 3h). We found that luciferase activity was approximately five-fold higher in IRF8-transduced cells compared with GFP-transduced cells (Fig. 3h). Together, these findings suggest that IRF5 is directly regulated by IRF8 in microglia.

**Figure 3: IRF5 expression is directly regulated by IRF8.**

We then asked whether IRF8 regulates the expression of IRF5 in microglia in vivo. We found that PNI-induced upregulation of IRF5 was inhibited in the spinal cord of mice lacking IRF8 (Irf8^−/−) both at the mRNA and protein level (Fig. 3i,j), indicating that the expression of IRF5 in spinal microglia is IRF8-dependent. The increase in the expression of spinal IRF8 after PNI was indistinguishable between WT and IRF5-deficient (Irf5^−/−) mice (Fig. 3k). Therefore, these results indicate that in spinal microglia, IRF5 is a downstream molecule of and is regulated by IRF8.

IRF8-mediated de novo expression of P2X4R requires IRF5

As forced expression of IRF8 in microglia has been shown to upregulate the expression of P2X4R (ref. 8), we hypothesized that IRF8-mediated P2X4R upregulation in microglia may be mediated by IRF5. To test this, we constructed viral vectors encoding IRF5 or control shRNA sequences expressed under an H1 promoter upstream of an EF-1α promoter-IRF8-GFP expression cassette (shIRF5-IRF8-GFP or shCtrl-IRF8-GFP, respectively), and performed gene transfer experiments (Fig. 4a). Cells transduced with shCtrl-IRF8-GFP showed increased expression of P2X4R and IRF5 compared with control cells (shCtrl-GFP) both at the mRNA and protein level (Fig. 4b–e). In contrast, the IRF8-mediated expression of P2X4R was markedly attenuated in cells with IRF5 knockdown (Fig. 4d,e), suggesting that IRF8-mediated de novo expression of P2X4R requires IRF5. Meanwhile, expression of Aif1 (encodes microglial Iba1), which is thought to be directly controlled by IRF8 (ref. 8), was not affected by IRF5 shRNA (Fig. 4f), indicating that knockdown of IRF5 does not result in a global impairment of IRF8-regulated gene expression. Collectively, IRF8-induced IRF5 expression may regulate the expression level of P2X4R in microglia.

**Figure 4: IRF8-mediated P2X4R upregulation in microglia depends on IRF5.**

IRF5 is required for upregulation of spinal P2X4R after PNI

We next investigated whether IRF5 was crucial for regulating the expression of P2X4R in microglia after PNI. Consistent with previous studies^8,32, the transcripts of P2rx4 were significantly increased 7 days after PNI in WT mice (Fig. 5a). In contrast, expression of P2rx4 was not altered in Irf5^−/− mice (Fig. 5a), emphasizing the importance of IRF5 for the upregulation of P2X4R in spinal microglia. P2X4R-stimulated microglia express and release BDNF, which is a crucial factor for producing tactile allodynia^13,14. Therefore, we determined the expression of BDNF in the spinal cord of Irf5^−/− mice. PNI increased the expression of Bdnf transcripts in the spinal cord of WT mice, whereas these changes were not observed in Irf5^−/− mice (Fig. 5a).

**Figure 5: IRF5 is required for *de novo* expression of P2X4R in the spinal cord after PNI but without microglial cellular alterations.**

IRF5 is reportedly critical for M1-related gene expression, including pro-inflammatory cytokines in macrophages (Supplementary Fig. 5)^17,18. Therefore, we further quantified transcripts of microglial genes related to inflammatory responses (IL-1β (Il1b), IL-6 (Il6), TNF-α (Tnfa), P2X7R (P2rx7), and cathepsin S (Ctss))^33,34,35. Surprisingly, Irf5^−/− mice showed comparable expressions of Il1b, Il6, Tnfa and P2rx7 to WT mice (Fig. 5a), and a slight inhibition of Ctss expression in Irf5^−/− mice was observed (Fig. 5a). Together, IRF5 deficiency specifically downregulates the expression of P2X4R and BDNF at the transcriptional level in spinal microglia after PNI.

IRF5-independent cellular alterations of microglia after PNI

In response to PNI, spinal microglia undergo morphological hypertrophy and proliferation^8,22. Iba1 staining revealed equivalent morphological alterations of spinal dorsal horn in Irf5^−/− compared with WT mice (Fig. 5b,c). Furthermore, immunofluorescence patterns of CD11b and CD68 (markers of activated microglia) were indistinguishable between the two genotypes (Fig. 5b). These expressional patterns of microglial markers were verified by real-time PCR analysis (Fig. 5d). These results indicate that PNI-induced morphological alterations of microglia in the spinal cord are independent of IRF5.

Tactile allodynia after PNI is dependent on IRF5

Our results suggest that the IRF8–IRF5 transcriptional network controls the expression of P2X4R in reactive microglia of the spinal dorsal horn after PNI. Microglial P2X4R signals are critical for generating neuropathic pain^12,13,35. Therefore, we next examined the functional relevance of IRF5 to tactile allodynia, a hallmark symptom of neuropathic pain characterized by abnormal pain hypersensitivity evoked by innocuous stimuli¹⁰. Allodynia was assessed by the paw withdrawal threshold (PWT) to mechanical stimulation of the hindpaw. In WT littermates (Irf5^+/+), PNI produced a robust and sustained reduction in PWT of the injured side (Fig. 6a). The decrease in PWT on day 1 was comparable between Irf5^−/− and Irf5^+/+ mice. After 3 days, PWT in Irf5^−/− mice was not further decreased (Fig. 6a). The behavioural difference between the two genotypes lasted until the final time point tested (day 14, Irf5^+/+: 0.275±0.040 g, Irf5^−/−: 0.966±0.117 g; Fig. 6a). In contrast, basal mechanical sensitivity or contralateral PWT remained unchanged in Irf5^−/− mice after PNI (Fig. 6a,b). Moreover, tail-flick and paw-flick (hot-plate tests) tests showed that acute physiological pain responses were the same between Irf5^+/+ and Irf5^−/− mice (Fig. 6e,f). These results exclude the possibility of congenital defects in pain processing in Irf5^−/− mice and also indicate that the deficiency of IRF5 does not affect general pain sensation. To further test the role of IRF5 in established pain hypersensitivity, we intrathecally administered small interfering RNA (siRNA) targeting IRF5 after PNI. This siRNA markedly suppressed the expression of IRF5 protein and ameliorated the established allodynia (Fig. 6c,d). An additional IRF5 siRNA also produced similar effects on pain behaviour (Supplementary Figs 6a, b and 15). These results indicate that continuous activity of microglial IRF5 in the spinal cord is required for chronic neuropathic allodynia after PNI.

**Figure 6: Loss of IRF5 abrogates PNI-induced tactile allodynia without affecting acute pain sensation or inflammatory pain.**

We also assessed behavioural responses in two different inflammatory pain models. Pain responses caused by injecting dilute formalin into the hindpaw (eliciting typical two-phase pattern of behavioural responses⁸) were indistinguishable between the two genotypes (Fig. 6g,h). Furthermore, intraplantar injection of complete Freund’s adjuvant (CFA), an established chronic inflammatory pain model⁸, caused a comparable reduction in PWT in both genotypes (Fig. 6i). In line with this result, expression of spinal IRF5 expression following CFA injection was unchanged (Supplementary Figs 6c and 15). These findings suggest that microglial IRF5 plays a role in the development and maintenance of chronic neuropathic pain without affecting acute physiological pain and local tissue inflammation-evoked pain.

Discussion

In the current study, we report a novel function of the IRF8–IRF5 transcriptional axis in shifting microglia toward the P2X4R⁺ phenotype, by which microglia play a crucial role in the pathogenesis of neuropathic pain (Fig. 7). To our knowledge, this report is the first to identify a microglia-specific transcription network that determines the distinct phenotypes of microglia.

**Figure 7: Schematic illustration and working models for determining P2X4R⁺-reactive microglia and neuropathic pain.**

Our detailed analyses reveal that following PNI, IRF5 is increased in spinal microglia in a cell-type-specific manner, depending on IRF8 through its direct binding to the promoter loci of IRF5 and activating the transcription of IRF5. Although the fact that the PNI-induced increased expression of IRF5 in the spinal cord was not completely eliminated in IRF8-deficient mice suggests that IRF8-mediated signals is not the only mechanism underlying upregulation of IRF5, our data strongly suggest that IRF8 acts as a crucial and dominant regulator of IRF5 expression.

Ectopic expression of IRF5 in cultured microglia did not induce any observable signs of microglial activation. Previous reports have shown that TLR signals activate gene expression via the IRF5 pathway in macrophages^17,18,19. However, under our experimental conditions, the TLR4 ligand, LPS, did not induce the translocation of microglial IRF5, which may be a result of a lower dependency of IRF5 on the TLR-mediated signalling pathway in microglia. This response corroborates with our findings that microglial gene expressions in response to TLR stimulation (via the TLR3 agonist Poly(I:C), or LPS) were only modestly changed by the knockdown of IRF5 (Supplementary Fig. 7). Previous studies have shown that fibronectin-mediated signals play a crucial role in the upregulation of P2X4R in microglia^26,29. Furthermore, fibronectin stimulation causes IRF5 to cluster on the putative promoter loci of P2X4R. Therefore, activation of IRF5 following fibronectin treatment in this study provided an interesting insight into a previously unknown function of microglial IRF5. We thus propose that IRF5 is an entirely novel transcription activator working at the promoter of P2X4R, thereby inducing de novo expression of P2X4R in microglia. The enhanced expression of IRF5 may allow microglia to elicit these responses more quickly and efficiently.

Our results also show that IRF5 deficiency affected IRF8-mediated upregulation of P2X4R, and yet the mechanism underlying this response remains unknown. Post-translational modifications, such as Lys63-linked polyubiquitination, have been considered as an important mechanism to control the localization or function of signalling molecules, including transcription factor³⁶. IRF5 harbours a consensus binding site for the ubiquitin ligase tumour necrosis factor receptor (TNFR)-associated factor 6 (TRAF6)^17,37, which is known to regulate Lys63-linked polyubiquitination of its substrates³⁸. Furthermore, IRF8 has an enhancing effect on TRAF6-induced ubiquitination³⁹. Therefore, in IRF8-transduced microglial cells, IRF5 may be activated via TRAF6-mediated polyubiquitination, resulting in enhanced expression of P2X4R in these cells. Although the molecular machineries of both fibronectin- and IRF8-mediated activation of IRF5 remain to be elucidated, our present data strongly suggest that the expression level of microglial P2X4R is concertedly regulated by IRF5.

Our results demonstrating an IRF8–IRF5–P2X4R axis in microglia may also raise the possibility that a PNI-associated signal that causes upregulation of IRF8 in spinal microglia results in inducing IRF5-mediated P2X4R expression. Although such signal remains to be identified, to determine whether the signal itself has a cooperative effect on IRF5-mediated P2X4R expression in microglia would be now a new important open question for future study.

Microglia have often been designed as M1 or M2 macrophage-like^40,41,42. Previous studies have shown that IRF5 is involved in the polarization of M1 macrophages, which exhibit progressive expressions of inflammatory molecules, such as pro-inflammatory cytokines¹⁸. However, gene expressions in the spinal cord of IRF5-deficient mice in the present study did not follow the expected patterns. Deficiency of IRF5 did not lead to a global defect in the expression of inflammatory components (for example, Il1b, Il6 and Tnfa), but affected the P2X4R–BDNF axis in the spinal cord in a restricted manner following PNI, though whether stimulating P2X4Rs is required for BDNF expression in microglia after PNI remains to be determined^13,15,16. Our data indicate that reactive spinal microglia after PNI cannot be classified into either reactive modality, but rather exhibit a neuropathic pain-specific signature. In addition, the function of IRF5 was found to be cell type- and location-dependent, which is consistent with previous reports^19,43.

Our previous study identified IRF8 as a key transcription regulator of microglia for its transformation into a reactive state and its involvement in the pathogenesis of neuropathic pain after PNI⁸. In the CNS parenchyma of Irf8^−/− mice, microglial cells exhibit an abnormal morphology without fine processes^44,45, by which microglia sense pathological alterations or disturbances to maintain homeostasis, suggesting that excessive repression of microglial IRF8 may inhibit the physiological functions of these cells. Therefore, defining transcriptional control mechanisms that participate or occur downstream of IRF8 and regulate specialized microglial genes (for example, P2X4R) involved in neuropathic pain may be more favourable for treating chronic pain conditions. Therefore, IRF5 may be a potential therapeutic target because our data reveal that Irf5^−/− mice exhibit less allodynic behaviour, with microglial morphology comparable to WT mice, which also suggests that PNI-induced morphological alteration of spinal microglia may not sufficiently contribute to the pathogenesis of neuropathic pain. Furthermore, our results demonstrating that suppressed spinal IRF5 by siRNA reverses pain hypersensitivity indicate that ongoing activity of microglial IRF5 is required for the development and maintenance of neuropathic pain, possibly through the P2X4R pathway.

In conclusion, this study provides a new insight into the mechanisms underlying the phenotypic shifts of microglia in response to injury or disease in the nervous system. Although the functions of microglial IRF5 in other CNS diseases is unknown, our results suggest a key transcriptional network that forms ‘neuropathic pain-driving microglia’, which can provide a potential therapeutic target.

Methods

Animals

Male IRF5-deficient mice (Irf5^−/−)¹⁷, IRF8-deficient mice (Irf8^−/−)⁴⁶ and their wild-type littermates, and C57BL/6 mice (Clea, Japan) were used. All mice used were aged 9–12 weeks at the start of each experiment, and were housed individually and in groups of two or three per cage at a temperature of 22±1 °C with a 12-h light–dark cycle, and were fed food and water ad libitum. All experimental procedures were performed under the guidelines of Kyushu University.

Microglial culture

Neonatal mouse brains were minced with a razor blade, and trypsinized in phosphate-buffered saline (PBS) containing 0.25% trypsin and 0.05% DNase at 37 °C. After adding fetal bovine serum, the dissociated cells were filtered and maintained in flasks for 10–16 days in DMEM containing 10% fetal bovine serum. Immediately before experiments, microglia were collected by a gentle shake as the floating cells over the mixed glial culture⁸. The microglia were transferred to dishes for subsequent experiments.

Peripheral nerve injury

We used the spinal nerve injury model⁴⁷ with some modifications⁸. Under isoflurane (2%) anaesthesia, a small incision at L3–S1 was made. Paraspinal muscle and fat were removed from the L5 transverse process, and the part of this transverse process was removed to expose the parallel-lying L3 and L4 spinal nerves, and then the L4 nerve was carefully isolated and cut. The wound and the surrounding skin were sutured with 5-0 silk.

Microarray analysis

Extraction of total RNA from the L3–L4 spinal cord using TRIsure (Bioline) and purification using RNeasy mini plus kits (Qiagen) were performed⁴⁸. Total RNA was converted to biotin-labelled cRNA, which was hybridized to the Mouse WG-6 V2.0 BeadChip (Illumina). Multiple gene expression in the spinal cord with PNI was analysed according to BeadStudio Gene Expression Module User Guide (Illumina).

Quantitative real-time PCR

Mice were deeply anaesthetized with pentobarbital, perfused transcardially with PBS and the L3–L4 spinal cord was removed immediately. The tissues were vertically separated by median, and hemisections of the spinal cord were subjected to total RNA extraction using Trisure (Bioline) according to the protocol of the manufacturer and purified with RNeasy mini plus kit (Qiagen, Valencia, CA, USA). Extraction of total RNA from primary cultured microglia or the microglial cell line BV-2 (provided by Dr Knut Biber, University of Freiburg) was also performed using TRIsure. The amount of total RNA was quantified by measuring OD260 using a Nanodrop spectrophotometer (Nanodrop, Wilmington, DE, USA). For reverse transcription, 150 ng of total RNA was transferred to the reaction with Prime Script reverse transcriptase (Takara). Quantitative PCR was performed with FastStart Essential DNA Probes Master (Roche) using a LightCycler 96, a LightCycler 480 system (Roche) or with Premix Ex Taq (Takara) using a 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). Expression levels were normalized to the values for 18s ribosomal RNA. The sequences of TaqMan primer pairs and probe are described below.

IRF5: 5′-CCTCAGCCGTACAAGATCTACGA-3′ (forward), 5′-GTAGCATTCTCTGGAGCTCTTCCT-3′ (reverse), 5′-FAM-CCAACGGCCCTGCTCCCACA-TAMRA-3′ (probe)

IRF8: 5′-GGATATGCCGCCTATGACACA-3′ (forward), 5′-CATCCGGCCCATACAACTTAG-3′ (reverse), 5′-FAM-CCATTCAGCTTTCTCCCAGATGGTCATC-TAMRA-3′ (probe)

Iba1(Aif1): 5′-GATTTGCAGGGAGGAAAAGCT-3′ (forward), 5′-AACCCCAAGTTTCTCCAGCAT-3′ (reverse), 5′-FAM-CAGGAAGAGAGGCTGGAGGGGATCAA-TAMRA-3′ (probe)

CD68: 5′-CTGCTGTGGAAATGCAAGCATA-3′ (forward), 5′-CCCGAAGTGTCCCTTGTCA-3′ (reverse), 5′-FAM-TCTCTCTAAGGCTACAGGCTGCTCAGCTGC-TAMRA-3′ (probe)

TLR2: 5′-CCCTTCTCCTGTTGATCTTGCT-3′ (forward), 5′-CGCCCACATCATTCTCAGGTA-3′ (reverse), 5′-FAM-CTGTGCCACCATTTCCACGGACTG -TAMRA-3′ (probe)

TLR4: 5′-AAACTTGCCTTCAAAACCTGGC-3′ (forward), 5′-ACCTGAACTCATCAATGGTCACATC-3′ (reverse), 5′-FAM-CACGTCCATCGGTTGATCTTGGGAGAA-TAMRA-3′ (probe)

CX3CR1: 5′-TCACCGTCATCAGCATCGA-3′ (forward), 5′-CTGCACTGTCCGGTTGTTCA-3′ (reverse),

5′-FAM-ATCGTCCTGGCCGCCAACTCC-TAMRA-3′ (probe)

P2X4R: 5′-ACAACGTGTCTCCTGGCTACAAT-3′ (forward), 5′-GTCAAACTTGCCAGCCTTTCC-3′ (reverse), 5′-FAM-CAATGAGCAACGCACACTCACCAAGG-TAMRA-3′ (probe)

P2X7R: 5′-TGCAGCTGGAACGATGTCTT-3′ (forward), 5′-CCAAAGCAAAGCTCTAATGTAGGAA-3′ (reverse), 5′-FAM-TATGAGACAAACAAAGTCACCCGGATCCA-TAMRA-3′ (probe)

P2Y12R: 5′-TGAAGACCACCAGGCCATTT-3′ (forward), 5′-AGGCCCAGATGACAACAGAAA-3′ (reverse), 5′-FAM-AAACGTCCAGCCCCAGCAATCTCTTG-TAMRA-3′ (probe)

IL-1β: 5′-GAAAGACGGCACACCCACC-3′ (forward), 5′-AGACAAACCGCTTTTCCATCTTC-3′ (reverse), 5′-FAM-TGCAGCTGGAGAGTGTGGATCCCAA-TAMRA-3′ (probe)

IL-6: 5′-GGGACTGATGCTGGTGACAA-3′ (forward), 5′-TGCCATTGCACAACTCTTTTCT-3′ (reverse), 5′-FAM-TCACAGAGGATACCACTCCCAACAGACCTG-TAMRA-3′ (probe)

BDNF: 5′-GCCCAACGAAGAAAACCATAAG-3′ (forward), 5′-TGTTTGCGGCATCCAGGTA-3′ (reverse), 5′-FAM-CACTTCCCGGGTGATGCTCAGCA-TAMRA-3′ (probe)

Cathepsin S (Ctss): 5′-TACATTCAGCTCCCGTTTGGT-3′ (forward), 5′-TCGTCATAGACACCGCTTTTGT-3′ (reverse), 5′-FAM-TCGACGCCAGCCATTCCTCCTTCT-TAMRA-3′ (probe), as well as the primers and probe for 18s were obtained from Applied Biosystems.

Western blotting

Cultured microglial cells were lysed in lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, protease inhibitors cocktail) and mixed with Laemmli sample buffer. For the mouse spinal cord homogenates, mice were deeply anaesthetized with pentobarbital, perfused transcardially with PBS, and the L3–L4 spinal cord was removed immediately. The tissues were vertically separated by median, and then hemisections of the spinal cord were homogenized in homogenization buffer (20 mM Tris–HCl (pH 7.4), 2 mM EDTA, 0.5 mM EGTA, 0.32 M sucrose, protease and phosphatase inhibitor cocktails) and centrifuged to remove cell debris. The supernatant was transferred to a new tube, mixed with Laemmli sample buffer and boiled at 95 °C. For isolation of nuclear and cytoplasmic protein, BV2 cells were lysed with buffer A (10 mM HEPES (pH 7.5), 10 mM KCl, 1.5 mM MgCl₂, 0.1 mM EGTA, 0.05% NP-40, 1.0 mM DTT, protease inhibitor cocktail), and then nuclei were recovered by centrifugation. The supernatant, which contained the cytoplasmic and membrane protein fractions, was collected for western blot analysis. Nuclear proteins were extracted by resuspending the nuclei pellet in buffer B (20 mM HEPES (pH 7.5), 25% (v/v) glycerol, 0.42 M NaCl, 1.0 mM EGTA, 1.0 mM EDTA, 1.0 mM DTT, protease inhibitor cocktail). After centrifugation, the supernatant that contained the nuclear protein fraction was collected. Aliquots (0.5–20 μg) were subjected to a 10% polyacrylamide gel electrophoresis, and proteins were transferred electrophoretically to PVDF membranes. After blocking with blocking one (Nacalai tesque), the membranes were incubated with anti-IRF5 rabbit polyclonal antibody (1:500, Abcam), anti-IRF8 goat polyclonal antibody (1:500, Santa Cruz Biotechnology), anti-P2X4R rabbit polyclonal antibody (1:1,000, Alomone), anti-β-actin mouse monoclonal antibody (1:2,000, Sigma), anti-α-tublin polyclonal antibody (1:1,000, Abcam), anti-lamin B polyclonal antibody (1:1,000, Santa Cruz) and then incubated with HRP-conjugated secondary antibody (1:1,000, GE Healthcare). The blots were detected using a chemiluminescence method (Chemi-Lumi One; Nacalai tesque) and exposed to films. Bands were quantified using the software NIH Image J 1.36, and the relative values of each protein were normalized by the values of the protein levels of β-actin, α-tublin or lamin B for the loading control.

ChIP-qPCR assay

ChIP assays were performed with SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology Japan, Tokyo, Japan) with anti-IRF1 (Santa Cruz Biotechnology), anti-IRF3(Santa Cruz Biotechnology), anti-IRF5 (Abcam), anti-IRF7 (Santa Cruz Biotechnology), anti-IRF8 (Santa Cruz Biotechnology), anti-IRF9 (Santa Cruz Biotechnology), normal goat IgG (Abcam) and normal rabbit IgG (Cell Signaling Technology) antibodies. ChIP signals were quantified by quantitative PCR analysis with a 7500 real-time PCR system (Applied Biosystems). Values obtained for immunoprecipitated samples (percent (%) input DNA) were normalized to values for respective normal IgG. The specific primer pairs for the Irf5 promoter region and P2rx4 promoter region, respectively, are described below.

Region 1: 5′-ATTTCTCAGGCCCTGTCTAAAGTG-3′ (forward),

5′-GGCACAGAGAGAGTTAGAGGAAGA-3′ (reverse)

Region 2: 5′-TATGGAGTCTTTCTGCACCCTGT-3′ (forward),

5′-TTCCAAGAACGAAGAGTCCCCTA-3′ (reverse)

ISRE-1: 5′-GCTGGCTCGTTTCAAGAATATT-3′ (forward),

5′-CGTACCCTGTAGCCGTCTATT-3′ (reverse)

ISRE-2: 5′-TCTACAGCCTGAAAGTCTATCATTG-3′ (forward),

5′-AAGGAATCTGAGAGGTACACACTG-3′ (reverse)

ISRE-3: 5′-GATAGGGAGAGGCTCGTTCA-3′ (forward),

5′-TAAAAGCTCGGGACCTGGAA-3′ (reverse)

ISRE-4: 5′-TACTGACCTGCCTCTTTTAAGGACA-3′ (forward),

5′-CGGAAAGAACTTTGAACCTTGAG-3′ (reverse)

Immunohistochemistry

Mice were deeply anaesthetized by pentobarbital and perfused transcardially with PBS followed by ice-cold 4% paraformaldehyde/PBS. The L4 segment of the lumbar spinal cord were removed, post-fixed in the same fixative and placed in 30% sucrose solution for 24 h at 4 °C. Transverse L4 spinal cord sections (30 μm) were incubated for 48 h at 4 °C with primary antibody. Identification of cell types was performed using the following markers: microglia, Cd11b (1:1,000, Serotec), Iba1 (ionized calcium-binding adapter molecule-1, 1:2,000, Wako) and Cd68 (1:1,000, Serotec); astrocytes, GFAP (1:500, Chemicon); neurons, NeuN (Neuronal Nuclei, 1:200, Chemicon). Spinal sections were incubated with secondary antibodies conjugated to Alexa Fluor 488 or 546 (1:1,000, Molecular Probes) and mounted in VECTASHIELD with or without DAPI (Vector Laboratories). Three to five sections from the L4 spinal cord segments of each mouse were randomly selected and analysed using an LSM510 Imaging System (Carl Zeiss, Japan).

Lentiviral transduction

The lentiviral CS2 vectors (RIKEN) encoding mouse IRF8–GFP, IRF8(K79E)–GFP, IRF5-GFP or GFP under the control of the human EF-1α promoter, or encoding IRF5 or control shRNA sequences expressed under the control of H1 promoter upstream of the EF-1α promoter–IRF8-GFP or GFP expression cassette were used. Each vector with pCAG-HIVgp (packaging plasmid; RIKEN), and pCMV-VSV-G-RSV-Rev (RIKEN) was cotransfected into HEK293T cells. After mixing with PEG, viral particles and 8 μg ml⁻¹ polybrene were added onto primary cultured microglia (1.2 × 10⁵ cells per well) or microglial BV2 cells (1.0 × 10⁴ cells per well) plated on 24-well plates⁴⁹. After a 12-hour treatment with the lentivirus, cultured medium was changed to conditioned medium prepared from the mixed glial culture, and microglia were further cultured for 60 h. For the gene expression experiments, the transduced microglia were subjected to total RNA extraction as described above.

Luciferase assay

To construct the Irf5 gene reporter plasmid, the 928-bp fragment from the Irf5 gene promoter was amplified by PCR from genomic DNA. The PCR products were subcloned into the region upstream of a luciferase expression cassette of the CS2 vector. Subsequently, viral particles carrying this cDNA were produced as stated above. Normal or IRF8-transduced BV2 cells were transduced with them. Then, the cells were plated, followed by lysing with Glo lysis buffer and the supernatant was assayed for luciferase activity with the Bright-Glo Luciferase Assay System (Promega).

Intrathecal injection of siRNA

Under isoflurane (2%) anaesthesia, mice were implanted with a 32-gauge intrathecal catheter (ReCathCo, Allison Park, PA, USA) through the atlanto-occipital region and in the lumbar enlargement (close to L3–L4 segments) of the spinal cord. After 7 days of recovery, the catheter placement was verified by the observation of transient hindpaw paralysis induced by intrathecal injection of lidocaine (2%, 1.5 μl). Animals that failed to show any paralysis were not used in experiments. Intrathecal injection of IRF5 siRNA or scrambled control siRNA (10 pmol per 2.5 μl) was followed by infusion of 3 μl of PBS. Sequences of the IRF5 siRNAs and the non-targeting scramble siRNA used in this study were as follows.

IRF5 siRNA-1 sense sequence: 5′-GCAGUUUAAAGAGCUUCAUUU-3′

IRF5 siRNA-1 antisense sequence: 5′-AUGAAGCUCUUUAAACUGCUU-3′.

IRF5 siRNA-2 sense sequence: 5′-GCCUAGAGCAGUUUCUCAAUU-3′.

IRF5 siRNA-2 antisense sequence: 5′-UUGAGAAACUGCUCUAGGCUU-3′.

Scramble siRNA sense sequence: 5′-GUUAGAAAGGGCAGAUAAAUU-3′.

Scramble siRNA antisense sequence: 5′-UUUAUCUGCCCUUUCUAACUU-3′.

Behavioural studies

To assess mechanical sensitivity, mice were placed individually in an opaque plastic cylinder, which was placed on a wire mesh and habituated for 1 h to allow acclimatization to the new environment. After that, calibrated von Frey filaments (0.02–2.0 g, North Coast Medical) were applied to the plantar surfaces of the hindpaws of mice with or without PNI, or peripheral inflammation caused by intraplantar injection of CFA (0.01 mg per 20 μl, Sigma), and the 50% PWT was determined using the up–down method⁵⁰. In a hot-plate test, mice were placed on a metal surface maintained at 45, 49 or 52 °C within a 25-cm-high Plexiglas box (25 cm × 2). The latency to either lick or shake the hindpaw or jump was measured as a nocifensive end point. Noxious heat-evoked tail flick responses were detected by the application of radiant heat (Ugo Basile, Italy) to the tail. The intensity of the heat stimulus was adjusted to 50 V, and the latency of the tail withdrawal response (seconds) was measured. In the tests of formalin-induced pain, mice were housed in individual box and allowed to habituate to the testing environment for 15 min. Then, mice were injected intraplantarly with formalin (5%, 20 μl), and then the duration of the licking and biting responses to the injected hindpaw was recorded at 5 min intervals for 60 min after the injection. All behavioural assessments were performed by an observer blind to experimental condition.

Statistical analysis

Statistical significance of differences was determined using the Student’s t test (Figs 3k and 6c,d), two-way ANOVA with post hoc Bonferroni test (Figs 1c,e and 6a), one-way ANOVA with a post hoc Dunnett’s multiple comparison test (Figs 3a and 6a,i), Bonferroni multiple comparison test (Figs 2c,e, 3b,c and 4b–f), or Tukey’s multiple comparison test (Figs 2b, 3f–h and 5a,d) using GraphPad Prism 5.04 software. Differences were considered significant at P<0.05.

Additional information

How to cite this article: Masuda, T. et al. Transcription factor IRF5 drives P2X4R⁺-reactive microglia gating neuropathic pain. Nat. Commun. 5:3771 doi: 10.1038/ncomms4771 (2014).

References

Hanisch, U. K. & Kettenmann, H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat. Neurosci. 10, 1387–1394 (2007).
Article CAS Google Scholar
Graeber, M. B. Changing face of microglia. Science 330, 783–788 (2010).
Article CAS ADS Google Scholar
Ransohoff, R. M. & Cardona, A. E. The myeloid cells of the central nervous system parenchyma. Nature 468, 253–262 (2010).
Article CAS ADS Google Scholar
Perry, V. H., Nicoll, J. A. & Holmes, C. Microglia in neurodegenerative disease. Nat. Rev. Neurol. 6, 193–201 (2010).
Article Google Scholar
Glass, C. K., Saijo, K., Winner, B., Marchetto, M. C. & Gage, F. H. Mechanisms underlying inflammation in neurodegeneration. Cell 140, 918–934 (2010).
Article CAS Google Scholar
Kierdorf, K. & Prinz, M. Factors regulating microglia activation. Front. Cell Neurosci. 7, 44 (2013).
Article CAS Google Scholar
Tsuda, M., Masuda, T., Tozaki-Saitoh, H. & Inoue, K. Microglial regulation of neuropathic pain. J. Pharmacol. Sci. 121, 89–94 (2013).
Article CAS Google Scholar
Masuda, T. et al. IRF8 is a critical transcription factor for transforming microglia into a reactive phenotype. Cell Rep. 1, 334–340 (2012).
Article CAS Google Scholar
McMahon, S. B. & Malcangio, M. Current challenges in glia-pain biology. Neuron 64, 46–54 (2009).
Article CAS Google Scholar
Tsuda, M., Inoue, K. & Salter, M. W. Neuropathic pain and spinal microglia: a big problem from molecules in ‘small’ glia. Trends Neurosci. 28, 101–107 (2005).
Article CAS Google Scholar
von Hehn, C. A., Baron, R. & Woolf, C. J. Deconstructing the neuropathic pain phenotype to reveal neural mechanisms. Neuron 73, 638–652 (2012).
Article CAS Google Scholar
Tsuda, M. et al. P2X4 receptors induced in spinal microglia gate tactile allodynia after nerve injury. Nature 424, 778–783 (2003).
Article CAS ADS Google Scholar
Beggs, S., Trang, T. & Salter, M. W. P2X4R+ microglia drive neuropathic pain. Nat. Neurosci. 15, 1068–1073 (2012).
Article CAS Google Scholar
Coull, J. A. et al. BDNF from microglia causes the shift in neuronal anion gradient underlying neuropathic pain. Nature 438, 1017–1021 (2005).
Article CAS ADS Google Scholar
Trang, T., Beggs, S. & Salter, M. W. ATP receptors gate microglia signaling in neuropathic pain. Exp. Neurol. 234, 354–361 (2012).
Article CAS Google Scholar
Ulmann, L. et al. Up-regulation of P2X4 receptors in spinal microglia after peripheral nerve injury mediates BDNF release and neuropathic pain. J. Neurosci. 28, 11263–11268 (2008).
Article CAS Google Scholar
Takaoka, A. et al. Integral role of IRF-5 in the gene induction programme activated by Toll-like receptors. Nature 434, 243–249 (2005).
Article CAS ADS Google Scholar
Krausgruber, T. et al. IRF5 promotes inflammatory macrophage polarization and TH1-TH17 responses. Nat. Immunol. 12, 231–238 (2011).
Article CAS Google Scholar
Tamura, T., Yanai, H., Savitsky, D. & Taniguchi, T. The IRF family transcription factors in immunity and oncogenesis. Annu. Rev. Immunol. 26, 535–584 (2008).
Article CAS Google Scholar
Negishi, H. et al. Cross-interference of RLR and TLR signaling pathways modulates antibacterial T cell responses. Nat. Immunol. 13, 659–666 (2012).
Article CAS Google Scholar
Rivest, S. Regulation of innate immune responses in the brain. Nat. Rev. Immunol. 9, 429–439 (2009).
Article CAS Google Scholar
Tsuda, M. et al. IFN-gamma receptor signaling mediates spinal microglia activation driving neuropathic pain. Proc. Natl Acad. Sci. USA 106, 8032–8037 (2009).
Article CAS ADS Google Scholar
Inoue, K. The function of microglia through purinergic receptors: neuropathic pain and cytokine release. Pharmacol. Ther. 109, 210–226 (2006).
Article CAS Google Scholar
Davalos, D. et al. ATP mediates rapid microglial response to local brain injury in vivo. Nat. Neurosci. 8, 752–758 (2005).
Article CAS Google Scholar
Nasu-Tada, K., Koizumi, S., Tsuda, M., Kunifusa, E. & Inoue, K. Possible involvement of increase in spinal fibronectin following peripheral nerve injury in upregulation of microglial P2X4, a key molecule for mechanical allodynia. Glia 53, 769–775 (2006).
Article Google Scholar
Tsuda, M. et al. Fibronectin/integrin system is involved in P2X(4) receptor upregulation in the spinal cord and neuropathic pain after nerve injury. Glia 56, 579–585 (2008).
Article Google Scholar
Price, J. & Hynes, R. O. Astrocytes in culture synthesize and secrete a variant form of fibronectin. J. Neurosci. 5, 2205–2211 (1985).
Article CAS Google Scholar
Echeverry, S., Shi, X. Q., Rivest, S. & Zhang, J. Peripheral nerve injury alters blood-spinal cord barrier functional and molecular integrity through a selective inflammatory pathway. J. Neurosci. 31, 10819–10828 (2011).
Article CAS Google Scholar
Tsuda, M., Toyomitsu, E., Kometani, M., Tozaki-Saitoh, H. & Inoue, K. Mechanisms underlying fibronectin-induced up-regulation of P2X4R expression in microglia: distinct roles of PI3K-Akt and MEK-ERK signalling pathways. J. Cell Mol. Med. 13, 3251–3259 (2009).
Article Google Scholar
Tamura, T., Nagamura-Inoue, T., Shmeltzer, Z., Kuwata, T. & Ozato, K. ICSBP directs bipotential myeloid progenitor cells to differentiate into mature macrophages. Immunity 13, 155–165 (2000).
Article CAS Google Scholar
Kurotaki, D. et al. Essential role of the IRF8-KLF4 transcription factor cascade in murine monocyte differentiation. Blood 121, 1839–1849 (2013).
Article CAS Google Scholar
Tsuda, M. et al. Lyn tyrosine kinase is required for P2X(4) receptor upregulation and neuropathic pain after peripheral nerve injury. Glia 56, 50–58 (2008).
Article Google Scholar
Clark, A. K. et al. Inhibition of spinal microglial cathepsin S for the reversal of neuropathic pain. Proc. Natl Acad. Sci. USA 104, 10655–10660 (2007).
Article CAS ADS Google Scholar
Burnstock, G. Purinergic signalling and disorders of the central nervous system. Nat. Rev. Drug Discov. 7, 575–590 (2008).
Article CAS Google Scholar
Inoue, K. & Tsuda, M. Microglia and neuropathic pain. Glia 57, 1469–1479 (2009).
Article Google Scholar
Chen, Z. J. & Sun, L. J. Nonproteolytic functions of ubiquitin in cell signaling. Mol. Cell 33, 275–286 (2009).
Article CAS Google Scholar
Balkhi, M. Y., Fitzgerald, K. A. & Pitha, P. M. Functional regulation of MyD88-activated interferon regulatory factor 5 by K63-linked polyubiquitination. Mol. Cell Biol. 28, 7296–7308 (2008).
Article CAS Google Scholar
Deng, L. et al. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell 103, 351–361 (2000).
Article CAS Google Scholar
Zhao, J. et al. IRF-8/interferon (IFN) consensus sequence-binding protein is involved in Toll-like receptor (TLR) signaling and contributes to the cross-talk between TLR and IFN-gamma signaling pathways. J. Biol. Chem. 281, 10073–10080 (2006).
Article CAS Google Scholar
Ransohoff, R. M. & Perry, V. H. Microglial physiology: unique stimuli, specialized responses. Annu. Rev. Immunol. 27, 119–145 (2009).
Article CAS Google Scholar
Starossom, S. C. et al. Galectin-1 deactivates classically activated microglia and protects from inflammation-induced neurodegeneration. Immunity 37, 249–263 (2012).
Article CAS Google Scholar
Miron, V. E. et al. M2 microglia and macrophages drive oligodendrocyte differentiation during CNS remyelination. Nat. Neurosci. 16, 1211–1218 (2013).
Article CAS Google Scholar
Couzinet, A. et al. A cell-type-specific requirement for IFN regulatory factor 5 (IRF5) in Fas-induced apoptosis. Proc. Natl Acad. Sci. USA 105, 2556–2561 (2008).
Article CAS ADS Google Scholar
Horiuchi, M. et al. Interferon regulatory factor 8/interferon consensus sequence binding protein is a critical transcription factor for the physiological phenotype of microglia. J. Neuroinflammation. 9, 227 (2012).
Article CAS Google Scholar
Minten, C., Terry, R., Deffrasnes, C., King, N. J. & Campbell, I. L. IFN regulatory factor 8 is a key constitutive determinant of the morphological and molecular properties of microglia in the CNS. PLoS One 7, e49851 (2012).
Article CAS ADS Google Scholar
Holtschke, T. et al. Immunodeficiency and chronic myelogenous leukemia-like syndrome in mice with a targeted mutation of the ICSBP gene. Cell 87, 307–317 (1996).
Article CAS Google Scholar
Kim, S. H. & Chung, J. M. An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 50, 355–363 (1992).
Article CAS Google Scholar
Tozaki-Saitoh, H. et al. P2Y12 receptors in spinal microglia are required for neuropathic pain after peripheral nerve injury. J. Neurosci. 28, 4949–4956 (2008).
Article CAS Google Scholar
Masuda, T., Tsuda, M., Tozaki-Saitoh, H. & Inoue, K. Lentiviral transduction of cultured microglia. Methods Mol. Biol. 1041, 63–67 (2013).
Article CAS Google Scholar
Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M. & Yaksh, T. L. Quantitative assessment of tactile allodynia in the rat paw. J. Neurosci. Methods 53, 55–63 (1994).
Article CAS Google Scholar

Download references

Acknowledgements

We thank Ms Nao Nishimoto for assisting with the experiments, Dr Keiko Ozato for kindly providing Irf8^−/− mice and Dr Tadatsugu Taniguchi for kindly providing Irf5^−/− mice. We also thank Dr Chie Hotta for assisting with the experiments. This work was supported by grants from the Japan Society for the Promotion of Science (JSPS) through the ‘Funding Program for Next Generation World-Leading Researchers (NEXT Program)’ initiated by the Council for Science and Technology Policy (CSTP) (M.T.) and the Ministry of Education, Culture, Sports, Science and Technology of Japan (T.M., M.T. and K.I.) and from the Japan Science and Technology Agency (JST) through the Core Research for Evolutional Science and Technology (CREST) program (K.I.), and was also supported by Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We appreciate the technical support from the Research Support Center, Graduate School of Medical Sciences, Kyushu University.

Author information

Authors and Affiliations

Department of Molecular and System Pharmacology, Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-ku, 812-8582, Fukuoka, Japan
Takahiro Masuda, Shosuke Iwamoto, Ryohei Yoshinaga, Hidetoshi Tozaki-Saitoh, Makoto Tsuda & Kazuhide Inoue
Core Research for Evolution Science and Technology, Japan Science and Technology Agency, Tokyo, 102-0076, Japan
Takahiro Masuda, Hidetoshi Tozaki-Saitoh & Kazuhide Inoue
Department of Immunology, Yokohama City University Graduate School of Medicine, 3-9 Fukuura, Kanazawa-ku, Yokohama, 236-0004, Japan
Akira Nishiyama & Tomohiko Tamura
Princess Margaret Cancer Centre, University Health Network, 620 University Avenue, Toronto, M5G 2C1, Ontario, Canada
Tak W. Mak

Authors

Takahiro Masuda
View author publications
You can also search for this author in PubMed Google Scholar
Shosuke Iwamoto
View author publications
You can also search for this author in PubMed Google Scholar
Ryohei Yoshinaga
View author publications
You can also search for this author in PubMed Google Scholar
Hidetoshi Tozaki-Saitoh
View author publications
You can also search for this author in PubMed Google Scholar
Akira Nishiyama
View author publications
You can also search for this author in PubMed Google Scholar
Tak W. Mak
View author publications
You can also search for this author in PubMed Google Scholar
Tomohiko Tamura
View author publications
You can also search for this author in PubMed Google Scholar
Makoto Tsuda
View author publications
You can also search for this author in PubMed Google Scholar
Kazuhide Inoue
View author publications
You can also search for this author in PubMed Google Scholar

Contributions

T.M. designed and performed most of the experiments, analysed the data, and wrote the manuscript. S.I. performed ChIP-qPCR assays and in vitro experiments. R.Y., H.T.-S. and A.N. assisted with the experiments. T.W.M. kindly provided Irf5^−/− mice. T.T. provided critical materials including Irf8^−/− mice and advice on data interpretation. M.T. conceived the study, supervised the overall project, designed the experiments and wrote the manuscript. K.I. supervised the overall project and wrote the manuscript.

Corresponding authors

Correspondence to Makoto Tsuda or Kazuhide Inoue.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1-15 (PDF 6194 kb)

Rights and permissions

This work is licensed under a Creative Commons Attribution 3.0 Unported License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/3.0/

Reprints and permissions

About this article

Cite this article

Masuda, T., Iwamoto, S., Yoshinaga, R. et al. Transcription factor IRF5 drives P2X4R⁺-reactive microglia gating neuropathic pain. Nat Commun 5, 3771 (2014). https://doi.org/10.1038/ncomms4771

Download citation

Received: 27 November 2013
Accepted: 01 April 2014
Published: 13 May 2014
DOI: https://doi.org/10.1038/ncomms4771

This article is cited by

Spinal P2X4 Receptors Involved in Visceral Hypersensitivity of Neonatal Maternal Separation Rats
- Ying Tang
- Li Chen
- Chun Lin
Purinergic Signalling (2023)
Neohesperidin Alleviates the Neuropathic Pain Behavior of Rats by Downregulating the P2X4 Receptor
- Yueying Wang
- Chenxi Li
- Shuangmei Liu
Neurochemical Research (2023)
BIN1 is a key regulator of proinflammatory and neurodegeneration-related activation in microglia
- Ari Sudwarts
- Supriya Ramesha
- Srikant Rangaraju
Molecular Neurodegeneration (2022)
Increased surface P2X4 receptors by mutant SOD1 proteins contribute to ALS pathogenesis in SOD1-G93A mice
- Eléonore Bertin
- Audrey Martinez
- Eric Boué-Grabot
Cellular and Molecular Life Sciences (2022)
MiR-106b-5p Attenuates Neuropathic Pain by Regulating the P2X4 Receptor in the Spinal Cord in Mice
- Huiying Du
- Danlei Wu
- Qingjuan Gong
Journal of Molecular Neuroscience (2022)

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.