Journal: Nature Communications

Article Title: Nlrp12 mutation causes C57BL/6J strain-specific defect in neutrophil recruitment

doi: 10.1038/ncomms13180

Figure Lengend Snippet: ( a ) WT (B6N) or Nlrp12 −/− mice were infected i.n. with 5 × 10 3 colony-forming unit of F. tularensis LVS. Three days post infection BAL were performed and cytokine levels were determined by ELISA ( n =8, WT; n =9, Nlrp12 −/−) . ( b ) BMDM from WT (B6N) and Nlrp12 −/− mice were challenged with either F. tularensis LVS, S. aureus or P. aeruginosa . Eight hours later supernatants were collected and secretion of the indicated cytokine or chemokine quantified by ELISA. ( c ) BMDM from WT (B6N) and Nlrp12 −/− mice were stimulated with LPS (50 ng ml −1 ) for the indicated amount of time; supernatants were collected and assayed for CXCL1 production by ELISA. ( d ) BMDM from WT (B6N) and Nlrp12 −/− mice were left unstimulated or stimulated for 4 h with LPS in the presence of brefeldin A and monensin; intracellular CXCL1 was then assessed by flow cytometry. ( e ) BMDM from WT (B6N) and Nlrp12 −/− mice were stimulated with LPS for the indicated amount of time; Cxcl1 expression was quantified by real-time PCR. ( f ) BMDM from WT (B6N), WT (B6J) and Nlrp12 −/− mice were stimulated with LPS for 8 h; supernatants were collected and assayed for the indicated cytokines by ELISA. ( g ) BMDM from B6N, B6J, B6N-B6J F1 and B6N-B6J F2 mice were challenged with LPS for 8 h; supernatants were collected and CXCL1 secretion assessed by ELISA. The Nlrp12 allele was sequenced for all B6N-B6J F2 mice and cohorts stratified based on their Nlrp12 genotype ( Nlrp12 B6N/B6N , Nlrp12 B6N/B6J or Nlrp12 B6J/B6J ) ( n =14, B6N; n =13, B6J; n =14, F1; n =52, F2). Pooled data from three ( b , c ) independent experiments are depicted or are representative of two ( d ) or three ( e , f ) independent experiments. ( b , c , e , f ) Data are expressed as the mean±s.e.m. * P <0.05, ** P <0.01, *** P <0.005, NS, not significant by Mann–Whitney U -test ( a , g ), Student's t -test ( b , e , f ) or two-way analysis of variance analysis ( c ).

Article Snippet: Antibody pairs for the IL-1β (MAB401 and BAF401; clone 30311 and polyclonal; R&D Systems; 8 and 4 μg ml −1 , respectively) and CXCL1 (mouse: MAB453 and BAF453; clone 48415 and polyclonal; R&D Systems; 8 and 0.4 μg ml −1 , respectively; human: MAB275, clone 20326, 4 μg ml −1 ; BAF275, polyclonal, 40 ng ml −1 ) and CCL3/ MIP-1α (Quantikine ELISA kit, Product #MMA00) ELISAs were from R&D Systems.

Techniques: Infection, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Expressing, Real-time Polymerase Chain Reaction, MANN-WHITNEY

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Cx43 is required for TNF-α-evoked and basal release of CXCL1 in astrocyte cultures. (A and B) CXCL1 release in astrocytes following TNF-α stimulation (10 ng/ml, 60 min). Note the TNF-α-induced CXCL1 release is suppressed by pretreatment (60 min) of CBX (20 and 100 µM, A) and Gap26 and Gap27 (100 µM, B) but not by the inhibitors of pannexin hemichannels probenecid (Prob, 500 µM, A) and PANX1 mimetic peptide 10Panx1 (100 µM, A) and the scrambled peptide (Gap27 scrambled, 100 µM, B). *P < 0.05, compared with control; #P < 0.05, compared with TNF-α. (C) Inhibition of basal release of CXCL1 by CBX in astrocytes. *P < 0.05, compared with control. (D) Evoked expression (content) of CXCL1 in astrocytes following TNF-α stimulation (10 ng/ml, 60 min). Note the TNF-α-induced CXCL1 expression is not suppressed by pretreatment (60 min) of CBX (20 and 100 µM) and inhibitors of pannexin hemichannels probenecid (Prob, 500 µM) and PANX1 mimetic peptide 10Panx1 (100 µM). In contrast, a high dose of CBX (100 µM) increases CXCL1 expression. *P < 0.05, compared with control; #P < 0.05, compared with TNF-α. (E) Effects of CBX on the basal expression (content) of CXCL1 in astrocytes. All data are mean ± SEM. n = 8 cultures/group. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: We also purchased SB 203580 from Calbiochem, SB 225002 from Tocris, CXCL1 neutralizing antibody from Boster, and normal Rabbit IgG from Santa Cruz.

Techniques: Control, Inhibition, Expressing

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Spinal injection of TNF-α-activated astrocytes induces mechanical allodynia via Cx43-mediated CXCL1 release. (A) Intrathecal injection of TNF-α-activated astrocytes elicited persistent mechanical allodynia for >48 h. Note this allodynia is reduced by pretreatment of astrocytes with Cx43 small interfering RNA (1 µg/ml, 18 h). *P < 0.05, compared with TNF-α or TNF-α + non-targeting control small interfering RNA treated group; n = 6 mice/group. (B) ELISA analysis shows increased CXCL1 release in the CSF at 3 h after the intrathecal injection of TNF-α-activated astrocytes. *P < 0.05, compared with vehcile group; #P < 0.05, compared with non-activated astrocytes; n = 4 mice/group. (C) Intrathecal injection of a CXCL1 neutralizing antibody (4 µg) transiently and partially reversed mechanical allodynia, induced by TNF-α-treated astrocytes. *P < 0.05, compared with control IgG group; n = 6 mice/group. (D) Intrathecal injection of the CXCR2 antagonist SB225002 (20 µg = 57 nmol) transiently and partially reversed mechanical allodynia, induced by TNF-α-activated astrocytes. *P < 0.05, compared with vehicle (PBS); n = 5–6 mice/group. All data are mean ± SEM. The differences between groups were analysed by ANOVA followed by Newman–Keuls test.

Article Snippet: We also purchased SB 203580 from Calbiochem, SB 225002 from Tocris, CXCL1 neutralizing antibody from Boster, and normal Rabbit IgG from Santa Cruz.

Techniques: Injection, Small Interfering RNA, Control, Enzyme-linked Immunosorbent Assay

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: CXCL1, upregulated in spinal cord astrocytes after nerve injury, enhances excitatory synaptic transmission in spinal cord neurons and maintains neuropathic pain via CXCR2. (A) Western blotting shows CXCL1 upregulation in the spinal cord dorsal horn 21 days after CCI. Right, quantification of Cx43 levels in the dorsal horn. The western blot results are presented as a fold of sham control. *P < 0.05, compared to sham control, Student’s t-test, n = 4 mice/group. (B) Intrathecal injection of SB 225002 (20 µg), 21 days after CCI, reduced CCI-induced mechanical allodynia in the late phase. *P < 0.05, compared with vehicle (saline), Student’s t-test, n = 6 mice/group. (C) Double immunostaining of CXCL1 and GFAP in the dorsal horn 21 days after CCI. Note CXCL1 is primarily colocalized with GFAP. Arrows indicate doubled-labelled cells. Scale bar = 50 µm. (D and E) CXCL1 superfusion (100 ng/ml) increases spontaneous EPSC frequency (revealed by patch clamp recordings) in lamina IIo neurons of spinal cord slices. (E) Spontaneous EPSC frequency. *P < 0.05, Student’s t-test, n = 5 neurons/group. (F and G) CCI (21 d) increases spontaneous EPSC frequency, which is reversed by the CXCR2 antagonist SB225002 (1 µM). *P < 0.05, Student’s t-test, n = 5 neurons/group.

Article Snippet: We also purchased SB 203580 from Calbiochem, SB 225002 from Tocris, CXCL1 neutralizing antibody from Boster, and normal Rabbit IgG from Santa Cruz.

Techniques: Transmission Assay, Western Blot, Control, Injection, Saline, Double Immunostaining, Patch Clamp

Journal: Brain

Article Title: Connexin-43 induces chemokine release from spinal cord astrocytes to maintain late-phase neuropathic pain in mice

doi: 10.1093/brain/awu140

Figure Lengend Snippet: Schematic of working hypothesis for astrocytic Cx43-mediated late-phase neuropathic pain. CCI induces a persistent upregulation of Cx43 in spinal cord astrocytes. Cx43 expression and activity is also upregulated by TNF-α, secreted from microglia. Upregulation of Cx43 hemichannel activities results in CXCL1 release. Astrocytic CXCL1 secretion activates CXCR2 on neurons (central terminals of primary sensory neurons and spinal cord neurons), leading to enhanced excitatory synaptic transmission in nociceptive neurons (e.g. lamina IIo excitatory interneurons) and sustained neuropathic pain in the late-phase. Additionally, CXCL1 can also be secreted from intact or injured primary afferents in the spinal cord especially in the early phase of CCI.

Article Snippet: We also purchased SB 203580 from Calbiochem, SB 225002 from Tocris, CXCL1 neutralizing antibody from Boster, and normal Rabbit IgG from Santa Cruz.

Techniques: Expressing, Activity Assay, Transmission Assay