Immunology and Cell Biology (2014) 92, 301–302 & 2014 Australasian Society for Immunology Inc. All rights reserved 0818-9641/14


Response to the Letter to the Editor Immunology and Cell Biology (2014) 92, 301–302; doi:10.1038/icb.2014.9

Progranulin (PGRN) has been reported by several, independent groups to have antiHowever, the inflammatory effects.1 molecular mechanism by which PGRN acts as an anti-inflammatory agent was first described by Liu’s group, who showed direct inhibition of TNF/TNFR1 (tumour necrosis factor/TNF receptor 1) signalling in their paper, Tang et al.,2 published in Science. In our work,3 we did not challenge the finding that PGRN is anti-inflammatory; however, we were unable to observe that PGRN has a direct effect on TNFR1 signalling as reported by Tang et al. Now Liu’s group have responded to our work and made certain claims regarding our findings, which we would like, in turn, to respond to. In their letter to the editor, Liu’s group claims that ‘PGRN-mediated inhibition of TNF activity has been well established’. This is inaccurate. In the nine articles cited, only three—Egashira et al.,4 Ve´zina et al.5 and Zhu et al.6—show PGRN inhibition of a TNFmediated effect. Three articles—Gou et al.,7 Kawase et al.8 and Thurner et al.9—show anti-inflammatory properties of PGRN in complex signalling scenarios where TNF has a role, but is not the only inflammatory mediator. Matsubara et al.10 reported a proinflammatory property of PGRN on adipocytes. None of these articles show binding of PGRN to TNFRs. The cited articles that address PGRN–TNFR interaction are from Liu’s group.11,12 On the other hand, Liu’s group does not cite work from two independent groups reporting on a comprehensive set of experiments that also, like us, fail to confirm that PGRN directly inhibits TNF signalling.13,14 A recurring topic in the interpretation of results concerns whether recombinant PGRN is properly folded. We acknowledged that we were unable to confirm whether our three sources of PGRN were correctly folded in the absence of a confirmed positive control for PGRN activity. Our sources included

recombinant mouse and human PGRN from Adipogen, and Liu’s group had previously reported that Adipogen was a reliable source of correctly folded PGRN (comment on Chen et al.13). In the current letter Liu’s group have refined their original claim and now state that one of five batches from Adipogen that they tested is inactive. We used two different batches of PGRN from Adipogen in our studies; therefore, the chances that we obtained at least one active batch is 96%. Liu’s group criticizes us for using improper experimental conditions in our study. They claim that we used a single dose of 250 ng ml 1 that was lower than they had shown was required to inhibit TNF signalling. This is not correct. In their Science paper, Tang et al. used a dose of 2.5 nM (B160 ng ml 1) in the same assays (Figure 6 in Tang et al.2) that we did (Figures 1a,b in Etemadi et al.3). They also state that ‘it is not clear whether PGRN itself affects the cell death and survival of U937 cells’. However, we showed that PGRN alone does not have any effect on survival of U937 cells and mouse dermal fibroblasts (Figures 1e,f in Etemadi et al.3). We tested PGRN/TNF ratios up to a 5000fold excess of PGRN. Liu et al. claim that despite the high ratio of PGRN to TNF, low concentrations of PGRN might be insufficient to saturate all receptors on the cell surface and the remaining free receptors might trigger downstream effects such as NF-kB and MAPK signalling. As discussed above, 250 ng ml 1 cannot be considered to be a low concentration and in Tang et al., they reported a higher affinity of PGRN to TNFR1 and TNFR2 than TNF to these receptors (Fig. 1b in Tang et al.2). In their letter Liu’s group provide new data and state that PGRN suppresses only 50% of TNF-induced genes in a whole-genome array. This new finding is inconsistent with their original claim that PGRN inhibits the very

first step in the signalling cascade, that is binding of the ligand to the receptor. In Etemadi et al., we concluded that ‘PGRN inhibition of TNF signalling is unlikely to be easy to observe with commonly available reagents and suggest that caution should be used before trying to develop reagents based on PGRN for clinical and preclinical studies.’ The recently published results that also fail to observe PGRN inhibition of TNF signalling but are not discussed in the response from Liu’s group obviously support our conclusion. The prospect of PGRN as an anti-inflammatory agent is indeed fascinating; however, the molecular mechanism needs to be revisited. CONFLICT OF INTEREST The authors declare no conflict of interest.

Nima Etemadi1,2, John Silke1,2 and Ueli Nachbur1,2 1CSCD

Division, The Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville, Victoria 3052, Australia and 2Department of Medical Biology, University of Melbourne, Parkville, Victoria 3052, Australia E-mail: [email protected]

1 Cenik B, Sephton CF, Kutluk CB, Herz J, Yu G. Progranulin: a proteolytically processed protein at the crossroads of inflammation and neurodegeneration. J Biol Chem 2012; 287: 32298–32306. 2 Tang W, Lu Y, Tian Q-Y, Zhang Y, Guo F-J, Liu G-Y et al. The growth factor progranulin binds to TNF receptors and is therapeutic against inflammatory arthritis in mice. Science 2011; 332: 478–484. 3 Etemadi N, Webb A, Bankovacki A, Silke J, Nachbur U. Progranulin does not inhibit TNF and lymphotoxinalpha signalling through TNF receptor 1. Immunol Cell Biol 2013; 91: 661–664. 4 Egashira Y, Suzuki Y, Azuma Y, Takagi T, Mishiro K, Sugitani S et al. The growth factor progranulin attenuates neuronal injury induced by cerebral ischemia-reperfusion through the suppression of neutrophil recruitment. J Neuroinflammation 2013; 10: 105.

Correspondence 302 5 Ve´zina A, Vaillancourt-Jean E, Albarao S, Annabi B. Mesenchymal stromal cell ciliogenesis is abrogated in response to tumor necrosis factor-a and requires NF-kB signaling. Cancer Lett 2014; 345: 100–105. 6 Zhu J, Nathan C, Jin W, Sim D, Ashcroft GS, Wahl SM et al. Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair. Cell 2002; 111: 867–878. 7 Guo Z, Li Q, Han Y, Liang Y, Xu Z, Ren T. Prevention of LPS-induced acute lung injury in mice by progranulin. Mediators Inflamm 2012; 2012: 540794. 8 Kawase R, Ohama T, Matsuyama A, Matsuwaki T, Okada T, Yamashita T et al. Deletion of progranulin exacerbates atherosclerosis in ApoE knockout mice. Cardiovasc Res 2013; 100: 125–133.

Immunology and Cell Biology

9 Thurner L, Zaks M, Preuss K-D, Fadle N, Regitz E, Ong MF et al. Progranulin antibodies entertain a proinflammatory environment in a subgroup of patients with psoriatic arthritis. Arthritis Res Ther 2013; 15: R211. 10 Matsubara T, Mita A, Minami K, Hosooka T, Kitazawa S, Takahashi K et al. PGRN is a key adipokine mediating high fat diet-induced insulin resistance and obesity through IL-6 in adipose tissue. Cell Metabolism 2012; 15: 38–50. 11 Jian J, Zhao S, Tian Q, Gonzalez-Gugel E, Mundra JJ, Uddin SMZ et al. Progranulin directly binds to the CRD2 and CRD3 of TNFR extracellular domains. FEBS Lett 2013; 587: 3428–3436.

12 Zhao Y-p, Tian Q-y, Liu C-j. Progranulin deficiency exaggerates, whereas progranulin-derived Atsttrin attenuates, severity of dermatitis in mice. FEBS Lett 2013; 587: 1805–1810. 13 Chen X, Chang J, Deng Q, Xu J, Nguyen TA, Martens LH et al. Progranulin Does Not Bind Tumor Necrosis Factor (TNF) Receptors and Is Not a Direct Regulator of TNF-Dependent Signaling or Bioactivity in Immune or Neuronal Cells. J Neurosci 2013; 33: 9202–9213. 14 Hu Y, Xiao H, Shi T, Oppenheim JJ, Chen X. Progranulin promotes TNF-induced proliferation of suppressive mouse CD4 þ Foxp3 þ regulatory T cells. Immunology (e-pub ahead of print 3 January 2014; doi:10.1111/ imm.12241).

Response to the letter to the editor.

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