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- 1. TRPM8 on mucosal sensory nerves regulates colitogenic responses by innate immune cells via CGRP Petrus R. de Jong, MD1,2,5, Naoki Takahashi, DDS, PhD1,3,5, Madusha Peiris, PhD4, Samuel Bertin, PhD1, Jihyung Lee, PhD1, Melanie G. Gareau, PhD1, Alan Paniagua, BS1, Alexandra R. Harris, MS1, David S. Herdman, BS1, Maripat Corr, MD, PhD1, L. Ashley Blackshaw, PhD4, and Eyal Raz, MD1,¶ 1Department of Medicine, University of California, San Diego, La Jolla, CA 92093, USA 2University Medical Center Utrecht, Utrecht, Lundlaan 6, 3508 GA Utrecht, The Netherlands 3Division of Oral Science for Health Promotion, Niigata University Graduate School of Medical and Dental Sciences, Niigata 951-8514, Japan 4Wingate Institute of Neurogastroenterology, Blizard Institute, Barts and The London School of Medicine and Dentistry, Queen Mary, University of London, London, UK Published in final edited form as: Mucosal Immunol. 2015 May ; 8(3): 491–504. doi:10.1038/mi.2014.82.
- 5. Bu çalışmanın çıkış noktası?? • Kolonda TRPV1+ / CGRP+ yoğun şekilde co-ekspresyonu görülmüş . TRPV1+ nöronlar dentritik hücrelerle oldukça yakın konumlanmış ve salgılanan CGRP nin antijen sunumunu ve migrasyonu downregüle edebildiği gösterilmiş (TLR4 ligandlarının aktivasyonu ve CGRP salınımı ve antiinflamatuar etkileri gösterilmiş) Mucosal Immunology (2014) 7, 1283–1289; doi:10.1038/mi.2014.80; published online 3 September 2014 Cross-talk between neural and immune receptors provides a potential mechanism of homeostatic regulation in the gut mucosa
- 6. • TRPA1 ve TRPV1 ile benzer fizyolojik karakter göstermekte ve yapılan bir çalışmada LPS ile sensitize edilmiş kanal TLR4 den bağımsız olarak CGRP ve P maddesi salınımı görülmüş • Merkezde CGRP olmak üzere TRPV1 ve TRPA1 bağırsak inflamasyonunda nöroimmun ve regulatuar prosesin majör komponenti olarak gösterilmiş • Bir çalışmada monositlerde TRPM2 nin artan ekspresyonu inflamatuar kapasiteyi arttırmış • Ramachandran et all. TRPM8 activation attenuates inflammatory responses in mouse models of colitis
- 8. AMAÇ • Kolonik TRPM8+ primer afferent nöronların, mukozal inflamatuar hücreler üzerinde lokal anti-inflamatuar etkileri olabilir mi?
- 9. Genetic deletion of TRPM8 increases the susceptibility to acute experimental colitis 1
- 10. TRPM8 deficiency does not affect chronic DSS or TNBS-induced colitisS1
- 11. TRPM8 deficiency does not affect chronic DSS or TNBS-induced colitis (devam) S1
- 12. Lack of epithelial traits predisposing for colitis in Trpm8-/- mice. S2
- 13. TRPM8 deficiency in non-hematopoietic cells results in a hyperinflammatory phenotype 2
- 14. TRPM8+ mucosal fibers in the colon express CGRP 3
- 15. TRPM8+ mucosal fibers in the colon express CGRP 3
- 16. CGRP receptor deficiency in hematopoietic cells confers susceptibility to colitis 4
- 17. CGRP receptor deficiency in hematopoietic cells confers susceptibility to colitis 5
- 18. CGRP treatment reverses the hyperinflammatory phenotype of TRPM8−/− mice 6
- 19. Working model of the effects of TRPM8+/CGRP+ mucosal fibers in colitis 7
- 20. Sonuçlar 1) Trpm8 -/- fareler DSS kolit modeline aşırı derecede duyarlıdır (şekil 1) 2) Trpm8 -/- fareler deki doğal bağışıklık hücreleri ex vivo olarak TLR ligandları ile stimüle edildiğinde hiperinflamatuar cevap sergilemektedir fakat bu durum myeloid hücrelerde içsel Trpm8 sinyali ile ilişkili değildir (şekil 2) 3) Fare ve insan kolonunda Trpm8+ dokuda CGPR-IR gözlenmiştir bunun yanısıra deneysel kolit modelinde mukozal dokuda da Trpm8/CGRP ekspresyon artışı gözlenmiştir (şekil 3). İnflame kolonda bu CGRP+ mukozal fiber CD11c+ hücrelere çok yakın olarak gözlenmiştir (şekil 4) 4) Trpm8 -/- fareler de DSS kolit modeli için bulunan aşırıduyarlılık CGPR sinyalindeki defekt ile fareler tarafından oluşan fenokopidir (Şekil 5) 5) Ekzojen CGRP ile tedavi Trpm8 -/- farelerin DSS aşırı duyarlı fenotipini geri çevirmiştir (şekil 6) 6) Lokal CGRP salınımına neden olan mukosal sensor nöronların alt kümesinde mukozal hasar ya da kolitin aktive ettiği Trpm8 sinyalini içeren bir model önerilmektedir.
- 21. Sonuçlar 7) CGRP nin lokal yüksek konsantrasyonu, doğal immun hücreler (CD11c+DC) tarafından üretilen pro-inflamatuar sitokinleri baskılamaktadır (Şekil 7a) 8) Bu immunoregulatör mekanizma; Trpm8 defektli farelerde mukozal fiber da CGRP artışına fakat dentritik hücrelere salgılanmamasına neden olup aşırı inflame fenotip ile sonuçlanmaktadır (şekil 7b) 9) Trpm8 -/- kolonda CGRP birikimi gözlemi ilginç bir gözlemdir bunun bu farelerde DSS impulsu sonrası gözlenen artmış inflamatuar aktividen kaynaklanabileceği düşünülmektedir. 10) Sonuç olarak; CGRP nin iyi yapılandırılmış anti-inflamatuar etkisi önerilen modelle uyumlu olabilir
- 23. Ek bilgi Intracellular noxious events following acid stimulation. Activation of the TRPV1 acid- sensing nociceptor induces Ca2+ influx causing activation of CaMK II by phosphorylation, followed by increased transcriptional activity of CREB, leading to up-regulation of CGRP production in DRG sensory neurons. CGRP is a neurotransmitter involved in nociceptive transmission via afferent sensory neurons and exacerbation of inflammation at peripheral sites via efferent sensory neurons.
- 24. LPS sensitizes the TLR4/TRPV1/CGRP pathway. Activation of TLR4 on macrophages and sensory nerves subsequently activates TRPV1 releasing CGRP. Anti-inflammatory effects of CGRP include indirect modulation of TNFα transcription via binding to CGRP receptor and upregulation of ICER (dotted line). LPS is shown as red dots. CGRP, sensory neuropeptide calcitonin gene-related peptide; ICER, inducible cyclic adenosine monophosphate early repressor; LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; TNFα, tumor necrosis factor-α; TRPV1, transient receptor potential cation channel subfamily V member 1. A full color version of this figure is available at the Mucosal Immunology journal online
Notes de l'éditeur
- Memeli transient receptör potansiyel iyon kanalı familyası toplam 28 üyeden oluşur, ve şekilde de görüldüğü gibi aminoasit sekanslarındaki homolojiye göre 6 subfamilyadan oluşur.
- Trpm8 kanalı 6 transmembran segmentten ve intrasellüler karboksi ve amino terminalinden oluşmuştur. S2 ve S3 mentol ve iicilin için bağlanma bölgesi S4 ve S5 voltajı sens eden bölge S5 ve S6 katyonlanların geçiş yaptığı bölegesi coiled-coil bölgesi olimerizasyondan sorumlu Trp domaini ise kanalın ısı sıcaklık bağımlı aktivasyonundan sorumlu Trpm kanalları 15-25 derece arası aktive oluyor ve ca geçişine izin veriyor (bunun mekanizması pip2 nin karboksi terminlinden pip2 ile interaksiyonu pip2 ve binding site ın afiniteside ısı düşmesi ile modüle ediliyor).aktivatörleri ph pıp2 endojen sinyal molekülleri Alternatif olarak cooling ajanlar vasıtasıyla örneğin mentol ve iicilin kanal aktive olabiliyor Negatif feedback makanizması ise pkc aracılıklı defosforilasyon Uyarılabilir olmayan hücrelerde, Ca2+ sinyalini oluşturan temel mekanizma, IP3 etkili Ca2+ salımıdır. Bu salımı sağlayan IP3 molekülü, hücre zarında bulunan fosfaditil inositol 4,5bifosfatın (PIP2), fosfolipaz C (PLC) tarafından IP3 ve diaçilgliserole (DAG) parçalanması ile oluşur. Başka bir sinyal molekülü olan DAG, hücrede önemli fonksiyonları olan protein kinaz C’yi (PKC) ve bazı TRP kanallarını aktive ederken, IP3, IP3R’lerden Ca2+ salımına yol açar.
- LPS sensitizes the TLR4/TRPV1/CGRP pathway. Activation of TLR4 on macrophages and sensory nerves subsequently activates TRPV1 releasing CGRP.18 Anti-inflammatory effects of CGRP include indirect modulation of TNFα transcription via binding to CGRP receptor and upregulation of ICER (dotted line). LPS is shown as red dots. CGRP, sensory neuropeptide calcitonin gene-related peptide; ICER, inducible cyclic adenosine monophosphate early repressor; LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; TNFα, tumor necrosis factor-α; TRPV1, transient receptor potential cation channel subfamily V member It is well established that dendritic cells sit in very close proximity to TRPV1+ neurons and that CGRP can downregulate antigen presentation and migration of dendritic cells in both the skin54, 63 and lung.64 Furthermore, CGRP has been shown to directly downregulate dendritic cell responses to TLR ligands.44 These studies suggest a host response to LPS that induces regulation; activation of TLR4 in the presence of TRPV1 ligands sensitizes TRPV1, lowering the threshold for activation, inducing release of CGRP, and promoting subsequent anti-inflammatory effects.65 CGRP therefore has the potential to provide host protection without compromising protective immune response
- LPS activates TLR4 that in turn initiates an intracellular cascade (TIRAP, MyD88, IRAK2, TRAF6) leading to the downstream activation of NF-κB and MAPK and the production of cytokines. LPS additionally activates PKCε through TIRAP and MyD88 that can potentially bind to TRPV1 via S800- and S502-binding sites. This results in the release of the modulatory neuropeptide CGRP.77 Other protein kinases have the ability to bind to TRPV1, i.e., PKA. In addition, Ca2+ (recruited through intracellular influx as a result of natural ligand binding to TRPV1) can activate PKCε for further modulation.
- LPS sensitizes the TLR4/TRPV1/CGRP pathway. Activation of TLR4 on macrophages and sensory nerves subsequently activates TRPV1 releasing CGRP.18 Anti-inflammatory effects of CGRP include indirect modulation of TNFα transcription via binding to CGRP receptor and upregulation of ICER (dotted line). LPS is shown as red dots. CGRP, sensory neuropeptide calcitonin gene-related peptide; ICER, inducible cyclic adenosine monophosphate early repressor; LPS, lipopolysaccharide; TLR4, Toll-like receptor 4; TNFα, tumor necrosis factor-α; TRPV1, transient receptor potential cation channel subfamily V member It is well established that dendritic cells sit in very close proximity to TRPV1+ neurons and that CGRP can downregulate antigen presentation and migration of dendritic cells in both the skin54, 63 and lung.64 Furthermore, CGRP has been shown to directly downregulate dendritic cell responses to TLR ligands.44 These studies suggest a host response to LPS that induces regulation; activation of TLR4 in the presence of TRPV1 ligands sensitizes TRPV1, lowering the threshold for activation, inducing release of CGRP, and promoting subsequent anti-inflammatory effects.65 CGRP therefore has the potential to provide host protection without compromising protective immune response
- Supplementary Figure S1. Trpm8-/- mice are hypersusceptible to acute but not chronic experimental colitis Supplementary Figure S1. Trpm8-/- mice are hypersusceptible to acute but not chronic experimental colitis. (a) Trpm8-/- mice showed shorter colons without pelleted contents after DSS treatment compared to WT mice. Typical examples are shown. (b) Quantification of colon lengths from WT and Trpm8-/- mice after DSS treatment. (c) Decreased expression of Muc2 and, (d) Muc3 transcripts in intestinal epithelial cells harvested from Trpm8-/- mice compared to WT, as determined by QPCR. Results were normalized to Gapdh. (e) Mean bodyweights of WT and Trpm8-/- mice during treatment with 3 cycles of DSS. Significant differences between groups were only observed during the first cycle i.e. the acute stage of colitis. (f) No statistically significant difference in DAI between WT and Trpm8-/- mice after 3 cycles of DSS. (g) The extent of mucosal injury and inflammatory cell infiltration were comparable between WT and Trpm8-/- mice after 3 cycles of DSS. (h) No difference in pro-inflammatory cytokine production between WT and Trpm8-/- mice after 3 cycles of DSS in colon explant cultures. (i) Similar or diminished levels of T-cell cytokines produced by MLN cells stimulated with anti-CD3/CD28 Abs in the Trpm8-/- cohort compared to WT. (j) Mean bodyweights of WT and Trpm8-/- mice after topical re-challenge with 2.5% (w/v) TNBS or EtOH (negative control) on day 0 after cutaneous sensitization with 1% (w/v) TNBS on day -7. No statistically significant differences were observed between the two cohorts throughout follow-up. (k) Reduced signs of colitogenic activity in colons from Trpm8-/- compared to WT mice after TNBS treatment. (l) No differences between WT and Trpm8-/- mice in innate (TNF-α, IL-1β and IL-6) or T-cell derived (IFNγ, IL-17A) pro-inflammatory cytokines in colon explant cultures after TNBS treatment. (m) Significantly reduced TNF-α production but Page 41 of 57 De Jong and Takahashi et al. TRPM8/CGRP+ fibres regulate mucosal inflammation 42 similar levels of IFNγ and IL-17A by anti-CD3/CD28 restimulated MLN cells isolated from Trpm8-/- mice compared to WT controls. Data are mean ± SEM. *P<0.05 by Student’s t-test (c-d, i, m) or ANOVA (e). Scale bars = 500 μm (g) or 250 μm (k).
- Supplementary Figure S2. Lack of epithelial traits predisposing for colitis in Trpm8-/- mice. (a) Similar expression levels of Muc2 and Muc3 in colonic epithelial cell lysates from naïve WT and Trpm8-/- mice, as determined by Q-PCR. Results were normalized for housekeeping gene Gapdh. (b) Comparable intestinal barrier functions measured in naïve WT and Trpm8-/- mice. Fecal albumin was measured by ELISA to quantify albumin leakage from the circulation to the intestinal lumen. (c) No difference in the uptake of dextran polymer FD-4 between WT and Trpm8-/- mice in vivo. FD-4 levels in the blood serum were quantified by measuring fluorescence, 2 hrs after oral gavage. (d) No differences in baseline active ion secretion (shortcircuit current, Isc), paracellular permeability (conductance, G) or macromolecular permeability (FITC dextran) as measured by Ussing chamber experiments with distal colon segments from naïve WT and Trpm8-/- mice. (e) No difference in the expression of proliferation marker Mki67 in intestinal epithelial cells between WT and Trpm8-/- mice. Mki67 expression was determined in intestinal epithelial cell lysates from the small intestine and colon, respectively, by Q-PCR (normalized for Gapdh). (f) No difference in the number of Ki67 positive cells in the small intestine or colon between WT and Trpm8-/- mice. Ki67 immunoreactivity in paraffin sections was detected with DAB (brown). Counterstaining was performed with hematoxylin (blue). Scale bar = 100 μm. (g) No expression of Trpm8 in intestinal epithelial cell lysates, as determined by Q-PCR. Positive control: spine homogenate. Neurofilament (neuronal marker). Villin (intestinal epithelial cell marker). Data are presented as mean ± SEM
- Figure 2. TRPM8 deficiency in non-hematopoietic cells results in a hyperinflammatory phenotype (a) Splenic DCs from Trpm8−/− mice are hyperinflammatory in response to TLR stimulation. CD11c+ DCs were isolated from the spleens from WT and Trpm8−/− mice, followed by stimulation with 10 ng/mL LPS or 5 μg/mL CpG, respectively. Supernatants were harvested 24 hrs after stimulation. TNF-α and IL-6 levels in supernatants were quantified by ELISA. *P<0.05 (t-test). (b) Lack of a hyperinflammatory phenotype of Trpm8−/− BMDCs. BMDCs were obtained by expanding bone marrow cells with 10 ng/mL GM-CSF. CD11c+ DCs were obtained by positive selection at day 6. BMDCs were then stimulated with 10 ng/mL LPS or 5 μg/mL CpG and supernatants were harvested 24 hrs later for pro-inflammatory cytokine detection. (c) No detection of Trpm8 transcripts in CD11c+ splenic DCs or BMDCs. Positive control: spinal cord homogenate. (d) Non-myeloid expression of TRPM8 determines the susceptible phenotype of Trpm8−/− mice in experimental colitis. BMC were performed as described in Methods. Six weeks after BM transplantation, mice were treated with DSS (2%) for 5 days and followed for bodyweight loss. Mice were euthanized on day 9 for determination of colitis severity. Trpm8−/− recipients showed an increased susceptibility to DSS colitis compared to WT hosts, independent of BM donor genotype, as shown by their body weight loss and DAI. *P<0.05, WT –> Trpm8 or Trpm8 –> Trpm8 vs. WT –> WT (ANOVA). (e) Enhanced TNF-α production in colon explant cultures by Trpm8−/− recipients compared to WT mice after DSS treatment. *P<0.05 vs. WT –> WT (ANOVA). (f) Increased mucosal damage and inflammatory cell infiltration in Trpm8−/− recipients compared to WT mice. Scale bar = 100 μm. Data are presented as mean ± SEM and representative of 3 independent experiments (a–c).
- Figure 3. TRPM8+ mucosal fibers in the colon express CGRP (a) Co-labeling for CGRP and TRPM8 in colon tissues from naïve WT mice. Representative pictures from IF staining showing TRPM8+/CGRP+ fibers in the mucosal layer (asterisk) and myenteric plexus (arrowheads). (b) Absence of TRPM8-IR in colons from Trpm8−/− mice. (c) In normal human ascending colon, CGRP-IR is found in fibers within the lamina propria, and in enteroendocrine (EE) cells lining the epithelium. TRPM8-IR is co-localised with CGRP only in fibers (asterisks). Right panel: interpretation of the merged IF image with TRPM8/CGRP co-labeling fibers in yellow. Representative results are shown (n=4). (d) Representative pictures of CGRP+ mucosal fibers in naïve and DSS-treated WT colons. Typically, naïve WT mice displayed an absence of CGRP-IR in the mucosa, which was markedly increased in inflamed colons (DSS). (e) Enhanced CGRP-IR was observed in naïve Trpm8−/− compared to WT mice, which was further increased after DSS treatment. (f) mice following disease, and when compared to WT DSS. (g) Representative picture of TRPM8/CGRP co-expression in mucosal fibers (asterisks) and myenteric plexus (arrowhead) after DSS treatment of WT mice. (h) Quantification of CGRP levels in colon homogenates (normalized per mg tissue) by ELISA, showing increased CGRP levels following DSS colitis in WT mice, with significantly elevated levels in naïve and DSS-treated Trpm8mice. (i) Co-labeling for TRPM8 and the epithelial marker, E-cadherin, of naïve and inflamed colon sections from WT mice. Enhanced TRPM8-IR was observed in the myenteric plexus (indicated by arrowheads) and mucosal fibers (asterisks) in DSS treated colons. Data are presented as mean ± SEM (f, h). *P<0.05 versus naïve WT, or as indicated (ANOVA). Imaging was performed at 63X (a–e, g) or 20X (i) magnification.
- Figure 4. Close proximity between CGRP+ mucosal fibers and CD11c+ DCs (a) CD11c+ cells were observed in the colonic lamina propria in close proximity to CGRP-IR+ fibers in DSS-treated WT and Trpm8−/− mice, respectively. Imaging was performed at 63X. (b) CD11c+ cells were significantly increased in numbers in DSS-treated Trpm8−/− compared to WT mice in colons, but not in spleens (n=4 for each group). Data are presented as mean ± SEM. *P<0.05 (t-test).
- Figure 7. Working model of the effects of TRPM8+/CGRP+ mucosal fibers in colitis (a) Proposed model of the local, anti-inflammatory effects of CGRP released by TRPM8+ sensory nerve fibers in the tissue microenvironment of the colon. In WT mice, the local release of CGRP by TRPM8+ mucosal fibers inhibits pro-inflammatory cytokine production by CD11c+ DCs in the course of mucosal damage and inflammation. (b) In Trpm8−/− mice, the lack of TRPM8 triggering results in CGRP accumulation in mucosal fibers. The absence of mucosal CGRP release results in unrestrained production of pro-inflammatory cytokines and enhanced colitis.