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• Re: Modulation of p38MAPK and STAT3 signalling by infliximab and etanercept in intestinal T-cells
  Antonio Di Sabatino
  Published on: 18 April 2016
• Modulation of p38MAPK and STAT3 signalling by infliximab and etanercept in intestinal T-cells
  Philip Rosenstiel
  Published on: 18 April 2016

• Published on: 18 April 2016

  Re: Modulation of p38MAPK and STAT3 signalling by infliximab and etanercept in intestinal T-cells

  • Antonio Di Sabatino, MD
  • Other Contributors:
    • Rachele Ciccocioppo, Raffaele Morera, and Gino R. Corazza

  Dear Editor

  We thank Rosenstiel et al for their interest in our paper regarding the role of the chimeric anti-tumor necrosis factor (TNF)-alpha antibody, infliximab, in reverting defective lamina propria T-cell apoptosis in Crohn’s disease via a caspase-dependent pathway.[1]

  In their interesting letter Rosenstiel et al investigate the complex TNF-alpha blocker machinery in Crohn’s disease...

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  Dear Editor

  We thank Rosenstiel et al for their interest in our paper regarding the role of the chimeric anti-tumor necrosis factor (TNF)-alpha antibody, infliximab, in reverting defective lamina propria T-cell apoptosis in Crohn’s disease via a caspase-dependent pathway.[1]

  In their interesting letter Rosenstiel et al investigate the complex TNF-alpha blocker machinery in Crohn’s disease by analyzing the influence of infliximab and recombinant TNF receptor/immunoglobulin G fusion protein, etanercept, on p38 mitogen-activated protein kinase (MAPK) activation, apoptosis and signal transducer and activator of transcription 3 (STAT3) tyrosine phosphorylation in lamina propria T-cells from active Crohn’s disease patients. Infliximab, but not etanercept, has been shown to result in p38MAPK activation and apoptosis, while both these agents were able to reduce STAT3 phosphorylation in Crohn’s disease lamina propria T-cells.

  If data on the differences in mucosal T-cell apoptosis-inducing capacity of infliximab and etanercept are in keeping with the results obtained by van den Brande et al,[2] the observation that infliximab induces activation of a pro-inflammatory MAPK does not tally with the evidence that the guanylhydrazone c-Jun N-terminal kinase/p38 inhibitor CNI-1493 induces clinical improvement in steroid-resistant Crohn’s disease patients.[3] The reason for this discrepancy probably lies in the complex and dichotomal role of p38MAPK in the inflammatory signal transduction. p38MAPK inhibition is efficacious in chronic inflammatory disease but not in acute experimental colitis, where it causes adverse effects [3,4] and it shares differential actions in different T-cell subpopulations [5]. In naïve T cells, in fact, the p38 inhibitor SB203580 inhibits the pro- inflammatory cytokine production, whereas in T helper type (Th) 1 cells it does not affect the TNF-alpha release but strongly impairs the anti- inflammatory IL-10 production, thus tilting the balance between Th1 and Th2 cytokines to the former.[5]

  Since several lines of evidence have also suggested the pro-apoptotic role of the p38 pathway,[6] Waetzig et al[5] hypothesized that the increased phosphorylation of the p38MAPK downstream effector ATF-2, found after infliximab treatment only in the responder Crohn’s disease patients, could enhance the infliximab-induced apoptosis of immune cells in this subgroup of patients. However, the similar proportion of lamina propria immune cell apoptosis after infliximab treatment in both responders and non responders suggests that differences in the signal transduction downstream of p38MAPK are not related to the infliximab-induced immune cell apoptosis.[5]

  Disturbances in STAT signaling pathways, which trasduce the immunomodulatory messages of most of cytokines/cytokine receptors, have been shown to be involved in the pathogenesis of Crohn’s disease. In particular, STAT3 and STAT4 proteins are constitutively activated in lamina propria CD4+ T-cells from active Crohn’s disease patients.[7] The finding shown by Rosenstiel et al that both infliximab and etanercept are able to reduce the in vitro STAT3 tyrosine phosphorylation of Crohn’s disease lamina propria T-cells is probably related to the capacity of both these agents to neutralize soluble TNF-alpha. Interestingly, the absence of quantitative differences in the potential of infliximab and etanercept in downregulating STAT3 signaling is consistent with the data of van den Brande et al,[2] who have shown that both drugs are able to neutralize soluble TNF-alpha to a similar extent.

  Taken together, these findings suggest that both the apoptotic and non-apoptotic intracellular signalling pathways underlying the therapeutic benefit of anti-TNF-alpha strategies in Crohn’s disease are multifacetted and more complex than initially thought. Dissecting the MAPK signaling cascades that are selectively activated in the abnormal immune response of inflammatory bowel disease is critical in the identification of selective targets and development of new and rational therapies for treatment of Crohn’s disease.

  References

  (1) Di Sabatino A, Ciccocioppo R, Cinque B, et al. Defective mucosal T cell death is sustainably reverted by infliximab in a caspase dependent pathway in Crohn’s disease. Gut 2004;53:70-7.

  (2) van den Brande JMH, Braat H, van den Brink GR, et al. Infliximab but not etanercept induces apoptosis in lamina propria T-lymphocytes from patients with Crohn’s disease. Gastroenterology 2003;124:1774-85.

  (3) Hommes D, van den Blink B, Plasse T, et al. Inhibition of stress- activated MAP kinases induces clinical improvement in moderate to severe Crohn’s disease. Gastroenterology 2002;122:7-14.

  (4) ten Hove T, van den Blink B, Pronk I, et al. Dichotomal role of inhibition of p38 MAPK with SB 203580 in experimetal colitis. Gut 2002;50:507-12.

  (5) Waetzig GH, Rosenstiel P, Nikolaus S, et al. Differential p38 mitogen-activated protein kinase target phosphorylation in responders and nonresponders to infliximab. Gastroenterology 2003;125:633-4; author reply 635-6.

  (6) Wada T, Penninger JM. Mitogen-activated protein kinases in apoptosis regulation. Oncogene 2004;23:2838-49.

  (7) Lovato P, Brender C, Agnholt J, et al. Constitutive STAT3 activation in intestinal T cells from patients with Crohn’s disease. J Biol Chem 2003;278:16777-81.

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  Conflict of Interest:
  None declared.

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• Published on: 18 April 2016

  Modulation of p38MAPK and STAT3 signalling by infliximab and etanercept in intestinal T-cells

  • Philip Rosenstiel, MD
  • Other Contributors:
    • Jorgen Agnholt, Jens Kelsen, Valentina Medici, Georg H. Waetzig, Dirk Seegert and Stefan Schreiber

  Dear Editor

  There is growing evidence that the efficacy of anti-TNF-a therapies in Crohn’s disease (CD) may critically depend on the binding of the transmembrane precursor of TNF-a (mTNF-a), thus eliciting complex intracellular signaling events, a process described as ‘reverse signaling’.[1-3]

  In their recent paper, Di Sabatino et al.[4] have shown that infliximab reverts defective peripheral and la...

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  Dear Editor

  There is growing evidence that the efficacy of anti-TNF-a therapies in Crohn’s disease (CD) may critically depend on the binding of the transmembrane precursor of TNF-a (mTNF-a), thus eliciting complex intracellular signaling events, a process described as ‘reverse signaling’.[1-3]

  In their recent paper, Di Sabatino et al.[4] have shown that infliximab reverts defective peripheral and lamina propria lymphocyte apoptosis in CD patients via a caspase-dependent mechanism, which further corroborates the findings of prior studies.[1-3,5] It has also been suggested that the failure of another TNF-binding agent, etanercept (Enbrel® ;a recombinant TNFR2:Fc fusion protein), to induce peripheral and lamina propria lymphocyte apoptosis,[5] provides a possible molecular explanation for the lack of efficacy of etanercept in a randomized, placebo-controlled trial in active CD.[6] However, the authors do not discuss other signaling pathways that are activated via the ligation of transmembrane TNF-a by infliximab, e.g. we have shown that infliximab also transiently activates p38 MAPK in monocytes in vitro and in the lamina propria of CD patients in vivo 3. Responders and non-responders to infliximab differ in the pattern of mucosal p38MAPK target phosphorylation, but not caspase-3 activation, further emphasizing the complex modulation of intracellular signaling pathways beyond the mere neutralization of sTNF-a.[7] To show if these signaling pathways are also activated in primary T-cells, we analyzed the influence of infliximab and etanercept on p38MAPK activation and apoptosis in an established model of non-transformed, in situ activated T- lymphocytes.[8,9]

  According to the findings of van den Brande et al,[5] we observed PARP cleavage as a molecular hallmark of apoptosis in cultures grown with infliximab (Fig.1A), but not in the presence of etanercept. Whereas no increase of phosphorylated p38MAPK could be detected after etanercept stimulation, a significant activation (i.e. dual phosphorylation) of p38 MAPK 24 h after infliximab treatment was observed in 4/5 of the cell lines derived from CD patients (Fig.1A).

  [View Figure 1 A and B]
  Figure 1

  The intestinal CD 4+ T cells were obtained from colonic biopsies from five patients with Crohn’s disease (median age 34, range 18-49) with an established diagnosis of Crohn’s disease according to histopathological and endoscopical criteria. All patients had active disease at inclusion treated with 5-ASA (2-4g/day, 5 patients), one patients was treated with prednisone (20mg/day) and four patients with azathioprine (150-200mg/day). None of the patients recieved anti-TNF-a treatment. Neither antigen nor feeder cells were added to the intestinal T cells cultures, which consisted of more than 97% CD3+ /CD4+ T cells.
  (A) Infliximab, but not etanercept activates p38a and induces apoptosis in intestinal T-lymphocytes from CD patients. Levels of (phosphorylated) p38 MAPK and PARP in in situ activated intestinal T-lymphocytes were investigated by Western blot 24h after the stimulation with the respective TNF-a binding agent as described 3. (n=5, data are representative of experiments performed in all patients)
  (B) STAT3 is activated in CD4+ cells in the intestinal mucosa in CD patients in vivo and downregulated by both infliximab and etanercept in intestinal T-cells from CD patients in vitro. Western blot and immunofluorescence staining were performed as described previously.[3] Filled arrowheads, cells positive for phospho-Y-STAT3 (anti-phospho-Y- STAT3, 1/100, Cell Signaling Technology) and CD4 (anti-CD4, 1/500, BD PharMingen, San Diego, CA); open arrowheads, CD4 immunoreactivity only (n=5 for immunofluorescence and Western blot, representative result for all experiments).

  We have demonstrated previously that constitutive tyrosine phosphorylation of the transcription factor signal transducer and activator of transcription 3 (STAT3) may represent a specific feature of intestinal T cells from Crohn's disease.[9] Tyrosine-phosphorylated STAT3 can be found in CD4+ cells in the inflamed mucosa as shown by immunofluorescence analysis. (Fig.1B). Surprisingly, infliximab and etanercept were able to reduce STAT3 phosphorylation in T-cells from CD patients (cultured with IL -2 and IL-4) to a similar extent (Fig.1B). It is tempting to speculate that the previously reported downregulation of IFN-g/GM-CSF by both infliximab and etanercept is also involved in the decrease of STAT3 tyrosine phosphorylation, as both cytokines are potent inducers of STAT3 activation via Janus kinases. Mechanistically, this common action of infliximab and etanercept might be due to the neutralization of an autocrine loop of constitutively released sTNF-a. Although the findings suggest that inhibition of cytokine-dependent, inducible STAT3 phosphorylation could be dispensable for therapeutic efficacy in CD, aberrant constitutive STAT phosphorylation in T-lymphocytes may still have an important modulatory role in chronic intestinal inflammation 9.

  These observations corroborate the hypothesis, that TNF-a binding agents may exert distinct functions on lymphocyte activation and survival, either by ‘reverse signaling’ via binding of mTNF-a resulting in p38MAPK activation and apoptosis or by neutralization of sTNF-a, which in this model may serve to inhibit STAT3 signaling. The individual properties of TNF-a blockers to induce either of these complex molecular actions may be differentially responsible for therapeutic success or failure in chronic inflammatory disorders such as rheumatoid arthritis, psoriasis or CD. In CD, the ligation of mTNF-a, subsequent apoptotic processes and MAPK signaling seems to be critically required. Molecular dissection of mTNF-a apoptotic and non-apoptotic signaling may have important implications for the design of future therapeutic strategies in IBD.

  References

  1. ten Hove T, van Montfrans C, Peppelenbosch MP, van Deventer SJ. Infliximab treatment induces apoptosis of lamina propria T lymphocytes in Crohn's disease. Gut 2002;50:206-11.

  2. Lugering A, Schmidt M, Lugering N, Pauels HG, Domschke W, Kucharzik T. Infliximab induces apoptosis in monocytes from patients with chronic active Crohn's disease by using a caspase-dependent pathway. Gastroenterology 2001;121:1145-57.

  3. Waetzig GH, Seegert D, Rosenstiel P, Nikolaus S, Schreiber S. p38 mitogen-activated protein kinase is activated and linked to TNF-alpha signaling in inflammatory bowel disease. J Immunol 2002;168:5342-51.

  4. Di Sabatino A, Ciccocioppo R, Cinque B, Millimaggi D, Morera R, Ricevuti L, Cifone MG, Corazza GR. Defective mucosal T cell death is sustainably reverted by infliximab in a caspase dependent pathway in Crohn's disease. Gut 2004;53:70-7.

  5. Van den Brande JM, Braat H, van den Brink GR, Versteeg HH, Bauer CA, Hoedemaeker I, van Montfrans C, Hommes DW, Peppelenbosch MP, van Deventer SJ. Infliximab but not etanercept induces apoptosis in lamina propria T- lymphocytes from patients with Crohn's disease. Gastroenterology 2003;124:1774-85.

  6. Sandborn WJ, Hanauer SB, Katz S, Safdi M, Wolf DG, Baerg RD, Tremaine WJ, Johnson T, Diehl NN, Zinsmeister AR. Etanercept for active Crohn's disease: a randomized, double-blind, placebo-controlled trial. Gastroenterology 2001;121:1088-94.

  7. Waetzig GH, Rosenstiel P, Nikolaus S, Seegert D, Schreiber S. Differential p38 mitogen-activated protein kinase target phosphorylation in responders and nonresponders to infliximab. Gastroenterology 2003;125:633-4; author reply 635-6.

  8. Agnholt J, Dahlerup JF, Kaltoft K. The effect of etanercept and infliximab on the production of tumour necrosis factor alpha, interferon- gamma and GM-CSF in in vivo activated intestinal T lymphocyte cultures. Cytokine 2003;23:76-85.

  9. Lovato P, Brender C, Agnholt J, Kelsen J, Kaltoft K, Svejgaard A, Eriksen KW, Woetmann A, Odum N. Constitutive STAT3 activation in intestinal T cells from patients with Crohn's disease. J Biol Chem 2003;278:16777-81.

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  Conflict of Interest:
  None declared.

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