Biological Activity of CXCL8 Forms Generated by Alternative Cleavage of the Signal Peptide or by Aminopeptidase-Mediated Truncation

Posttranslational modification of chemokines is one of the mechanisms that regulate leukocyte migration during inflammation. Multiple natural NH(2)-terminally truncated forms of the major human neutrophil attractant interleukin-8 or CXCL8 have been identified. Although differential activity was reported for some CXCL8 forms, no biological data are available for others.status: publishe

CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice.

Neuroinflammation and microglial activation are significant processes in Alzheimer's disease pathology. Recent genome-wide association studies have highlighted multiple immune-related genes in association with Alzheimer's disease, and experimental data have demonstrated microglial proliferation as a significant component of the neuropathology. In this study, we tested the efficacy of the selective CSF1R inhibitor JNJ-40346527 (JNJ-527) in the P301S mouse tauopathy model. We first demonstrated the anti-proliferative effects of JNJ-527 on microglia in the ME7 prion model, and its impact on the inflammatory profile, and provided potential CNS biomarkers for clinical investigation with the compound, including pharmacokinetic/pharmacodynamics and efficacy assessment by TSPO autoradiography and CSF proteomics. Then, we showed for the first time that blockade of microglial proliferation and modification of microglial phenotype leads to an attenuation of tau-induced neurodegeneration and results in functional improvement in P301S mice. Overall, this work strongly supports the potential for inhibition of CSF1R as a target for the treatment of Alzheimer's disease and other tau-mediated neurodegenerative diseases.Funded by a grant from the Wellcome Trust (Grant number: 104025/Z/14/Z), and by the NIHR Oxford Health Biomedical Research Centre

CSF1R inhibitor JNJ-40346527 attenuates microglial proliferation and neurodegeneration in P301S mice

Neuroinflammation and microglial activation are significant processes in Alzheimer’s disease pathology. Recent genome-wide association studies have highlighted multiple immune-related genes in association with Alzheimer’s disease, and experimental data have demonstrated microglial proliferation as a significant component of the neuropathology. In this study, we tested the efficacy of the selective CSF1R inhibitor JNJ-40346527 (JNJ-527) in the P301S mouse tauopathy model. We first demonstrated the anti-proliferative effects of JNJ-527 on microglia in the ME7 prion model, and its impact on the inflammatory profile, and provided potential CNS biomarkers for clinical investigation with the compound, including pharmacokinetic/pharmacodynamics and efficacy assessment by TSPO autoradiography and CSF proteomics. Then, we showed for the first time that blockade of microglial proliferation and modification of microglial phenotype leads to an attenuation of tau-induced neurodegeneration and results in functional improvement in P301S mice. Overall, this work strongly supports the potential for inhibition of CSF1R as a target for the treatment of Alzheimer’s disease and other tau-mediated neurodegenerative diseases

Citrullination of TNF-α by peptidylarginine deiminases reduces its capacity to stimulate the production of inflammatory chemokines

Citrullination, a posttranslational modification (PTM) recently discovered on inflammatory chemokines such as interleukin-8 (IL-8/CXCL8) and interferon-γ-inducible protein-10 (IP-10/CXCL10), seriously influences their biological activity. Citrullination or the deimination of arginine to citrulline is dependent on peptidylarginine deiminases (PADs) and has been linked to autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA). Chemokines are to date the first identified PAD substrates with receptor-mediated biological activity. We investigated whether cytokines that play a crucial role in RA, like interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF-α), may be citrullinated by PAD and whether such a PTM influences the biological activity of these cytokines. IL-1β and TNF-α were first incubated with PAD in vitro and the occurrence of citrullination was examined by Edman degradation and a recently developed detection method for citrullinated proteins. Both techniques confirmed that human TNF-α, but not IL-1β, was citrullinated by PAD. Citrullination of TNF-α reduced its potency to stimulate chemokine production in vitro on human primary fibroblasts. Concentrations of the inflammatory chemokines CXCL8, CXCL10 and monocyte chemotactic protein-1 (MCP-1/CCL2) were significantly lower in supernatants of fibroblasts induced with citrullinated TNF-α compared to unmodified TNF-α. However, upon citrullination TNF-α retained its capacity to induce apoptosis/necrosis of mononuclear cells, its binding potency to Infliximab and its ability to recruit neutrophils to the peritoneal cavity of mice.status: publishe

Susceptibility of CXCL8(-2-77) to proteolysis by plasmin.

<p>CXCL8(1-77) and CXCL8(-2-77) were incubated with plasmin and the degree of conversion was determined using electrospray ion trap mass spectrometry. Percentages of intact CXCL8 [either CXCL8(1-77) or CXCL8(-2-77)] (black histograms), CXCL8(6-77) (dark grey histograms) and CXCL8(9-77) (light grey histograms) generated upon incubation with plasmin are depicted in function of time (min).</p

Chemokine receptor-dependent activity of the elongated CXCL8 variant <i>in vitro</i>.

<p>Intracellular calcium signaling upon activation of CXCR1 or CXCR2 was measured in HEK293 cells transfected with CXCR1 (panel A) or CXCR2 (panel B) or in neutrophilic granulocytes (panel C). Increases of the [Ca²⁺]_i (± SEM) are shown upon stimulation of the cells with the indicated concentrations of CXCL8(1-77) (♦) or CXCL8(-2-77) (○). The dashed line indicates the detection limit (an increase in [Ca²⁺]_i of 20 nM). The Mann-Whitney U test was used to detect statistical differences [*, p<0.05; compared to the corresponding concentration of CXCL8(1-77)] (n≥3). The neutrophil chemotactic activity of CXCL8(1-77) (♦) or CXCL8(-2-77) (○) was evaluated in 96-well Boyden chambers (panel D). Values represent the mean chemotactic index (± SEM). Statistical analysis was performed using the Mann-Whitney U test [*, p<0.05; compared to the corresponding concentration of CXCL8(1-77)] (n = 6).</p

Overview of the naturally occurring CXCL8 isoforms.

<p>Several NH₂-terminally modified isoforms of CXCL8 have been purified from the conditioned medium of leukocytes, fibroblasts and endothelial cells. The NH₂-terminal and COOH-terminal sequences of these isoforms are depicted in the one-letter code (B = citrulline). Based on the criteria formulated by Von Heijne <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0023913#pone.0023913-vonHeijne1" target="_blank">[29]</a>, the 99 amino acid precursor molecule may be cleaved by signal peptidases between Cys₂₀ and Glu₂₁ or between Gly₂₂ and Ala₂₃, the latter being more probable. Indeed both resulting CXCL8 proteins have been identified and are indicated in this manuscript as CXCL8(-2-77) and CXCL8(1-77), respectively. CXCL8(1-77) can be cleaved by a number of proteases, resulting in the listed NH₂-terminally truncated forms. Furthermore, CXCL8 can be citrullinated by peptidylarginine deiminase (PAD). These NH₂-terminal modifications differently affect the ability of CXCL8 to recruit neutrophils. The <i>in vivo</i> neutrophil-attracting activity is defined in the boxes at the right. CXCL8(-2-77), CXCL8(1-77), CXCL8(2-77) and CXCL8(3-77) show comparable intermediate activity. The more extensively truncated isoforms CXCL8(6-77), CXCL8(7-77), CXCL(8-77) and CXCL8(9-77), in contrast, display enhanced neutrophil recruiting potency, whereas citrullination significantly lowers the capacity of CXCL8 to guide neutrophils. (MMP, matrix metalloprotease).</p

Neutrophil-attracting activity of the CXCL8 isoforms <i>in vivo</i>.

<p>The neutrophil influx upon i.p. injection of the specified doses of the CXCL8 isoforms in mice was investigated. 2 hours post-injection, the peritoneal cavity was washed and the overall number of leukocytes determined. Differential microscopic counting of Hemacolor-stained cells allowed to define the percentage of neutrophils (panel A) and the total number of neutrophils (panel B) present in the peritoneal cavity. Squares show the median neutrophil influx (formulated as percentages or total counts/ml); the bottom and the top of the rectangle denote the 25th and 75th percentile; whiskers represent the non-outlier range (coefficient 1.5). Outliers are plotted as circles. Control mice (co) were treated with vehicle and illustrate the spontaneous migration of neutrophils to the peritoneal cavity. The Mann-Whitney U test was used for statistical analysis [§, p<0.01; compared with 100 pmol CXCL8(1-77)]. The numbers depicted in (or besides) the rectangular box represent the number of mice treated.</p

Chemokine receptor-dependent activity of the truncated CXCL8 isoforms <i>in vitro</i>.

<p>Intracellular calcium signaling upon activation of CXCR1 or CXCR2 was measured in HEK293 cells transfected with CXCR1 (panel A) or CXCR2 (panel B) or in neutrophilic granulocytes (panel C). Increases in the [Ca²⁺]_i (± SEM) are shown upon stimulation of the cells with the indicated concentrations of CXCL8(1-77) (♦), CXCL8(2-77) (▪), CXCL8(3-77) (▴) or CXCL8(6-77) (•). The dashed line indicates the detection limit (an increase in [Ca²⁺]_i of 20 nM). The Mann-Whitney U test was used to detect statistical differences [*, p<0.05; **, p<0.01; compared to the corresponding concentration of CXCL8(1-77)] (n≥3). The neutrophil chemotactic activity of CXCL8(1-77) (♦), CXCL8(2-77) (▪), CXCL8(3-77) (▴) and CXCL8(6-77) (•) was evaluated in 96-well Boyden chambers (panel D). Values represent the mean chemotactic index (± SEM). Statistical analysis was performed using the Mann-Whitney U test [*, p<0.05; **, p<0.01; compared to the corresponding concentration of CXCL8(1-77)] (n = 6).</p