Interleukin-6 Receptor Signaling and Abdominal Aortic Aneurysm Growth Rates

Supplemental Digital Content is available in the text.


Introduction
Abdominal aortic aneurysms (AAAs) are defined as an enlargement of the aorta to ≥30mm diameter. They usually grow asymptomatically until rupture occurs, after which the survival of affected individuals is less than 20%. 1 AAAs typically occur in mid-to-later life and more commonly in men (prevalence of 1-2% 2, 3 compared to <1% in women 4 ). Current standard of care is surgical intervention, either open surgery or endovascular repair. However, due to surgical risks vs. benefits, such interventions are generally recommended only for people with larger AAAs (diameter 55mm or >40mm and enlarging >10mm/year). 5,6 The growth rates of AAAs vary considerably between individuals 7 and there is currently a lack of therapeutic options to slow or halt progression of AAAs. 8 Inflammatory processes in the vessel wall may contribute to the progression of AAAs. [9][10][11] For example, levels of circulating inflammatory markers including interleukin (IL)-6 are higher in prevalent AAA cases than controls 12 and correlate with the size of AAA in cross-sectional studies. 13 IL-6 is a central coordinator of inflammatory responses by controlling the systemic inflammatory response in the liver and the activation and differentiation of leukocyte subsets, including macrophages and T cells. IL-6 signalling occurs in two different modes, termed "classical" (cis-) and trans-signalling. In classical signalling, binding of IL-6 to the membranebound IL-6 receptor (mIL-6R) induces homodimerization with its co-receptor gp130, resulting in the phosphorylation of the transcription factors STAT3 and STAT1. 14,15 Hence, classical signalling is dependent on the membrane-bound form of the IL-6 receptor and occurs only in leukocyte subsets and hepatocytes that express this molecule. By contrast, trans-signalling occurs through a circulating soluble form of IL-6R (sIL-6R), which, if bound to IL-6, is able to stimulate cells expressing gp130, even in the absence of mIL-6R. 14,16 Because gp130 is almost ubiquitously expressed, IL-6 trans-signalling can occur in virtually any cell, although is probably active only during conditions of immunological stress. 14,16 A non-synonymous variant (Asp358Ala; rs2228145 A>C) in the IL6R gene, encoding the IL-6 receptor, plays a critical role in IL-6 signalling. The minor allele [C] of this variant is associated with a reduced risk of several chronic conditions including coronary heart disease, 17 atrial fibrillation, 18 rheumatoid arthritis, 19 type 1 diabetes, 20 but an increased risk of asthma. 21 The variant results in more efficient proteolytic cleavage of mIL-6R, thereby reducing levels of mIL-6R and dampening classical signalling. 22,23 Conversely, the variant increases levels of sIL-6R, although the exact effects of the variant on the trans-signalling pathway are unknown. 20 A Mendelian randomisation study has previously implicated the rs2228145 in the causal pathway of AAA, with the minor allele [C] showing a protective effect for the risk of AAA and a combined endpoint of rupture or surgical intervention. 12 Licenced drugs are available that target the IL-6/IL-6 receptor pathway. However, evidence is needed that this pathway is associated with aneurysm progression or rupture in order to encourage repurposing drugs for use in patients with known AAA.
The aims of this study were to: (1) to assess and quantify the effect of the functional IL6R variant on the progression of AAAs in population cohorts with prospective follow-up and standardized repeated measurements of AAA diameter; and (2) estimate the effect of blocking the IL-6 signalling pathway (i.e., either both classical and trans-signalling pathways or specifically the trans-signalling pathway) on time to aneurysm rupture in mouse models.

Methods
Details on the materials and methods are available in the Supplemental Material. Because of the sensitive nature of the data collected for this study, requests to access the human AAA datasets from qualified researchers trained in human subject confidentiality protocols may be sent to the study leaders of each cohort. The experimental data that support the findings of this study are available from the corresponding author upon reasonable request. Each human AAA cohort was approved by a research ethics committee and all participants gave informed consent. Animal experiments were approved by the UK Home Office and performed under PPL PA4BDF775.
The care and use of all mice in this study was carried out in accordance with the UK Home Office regulations under the Animals (Scientific Procedures) Act 1986.

Association of IL6R-Asp358Ala with AAA growth rate
We studied a total of 2,863 participants across nine prospective cohorts, 91% of whom were men (mean age of 72 years; Table 1). AAA size was on average 43mm at baseline and participants had an average of four scans across an average of three years (Table 1). A summary of baseline characteristics by IL6R-rs2228145 (or proxy) genotype is given in Supplementary Table 1 [ Table 1]

Inhibition of IL-6 signalling pathway in angiotensin II + anti-TGFβ mouse model
We next tested the effect of blocking the IL-6 pathway in two distinct, previously characterised mouse models of AAA (Methods). In the angiotensin II + anti-TGFβ model, mice infused with anti-IL-6R (blocking both classical and trans-signalling pathways) demonstrated a significant increase of plasma concentration of IL-6, as compared to isotype treated mice, and this difference was sustained over the course of the experiment (Figure 2A). We observed a reduction in plasma concentrations of serum amyloid A (SAA), a protein expressed in response to inflammation, after blocking IL-6R compared to the control mice ( Figure 2B). Blocking IL-6R significantly reduced plasma concentration of IL-2 before and after the infusion and reduced concentration of IL-5 and chemokine ligand 1 (CXCL1) after the infusion (Figures 2A). After anti-IL-6R treatment, systolic blood pressure was significantly lower after the infusion compared to control treatment ( Figure 2C). However, there was no significant difference in rate of aneurysm rupture between the anti-IL-6R treated and control groups ( Figure 2D). Since there was no observed association with AAA rupture, we did not further assess the effect of blocking the IL-6R pathway on AAA growth.
Selectively blocking the IL-6 trans-signalling pathway using sgp130Fc did not change the concentration of IL-6 ( Figure 3A) or SAA ( Figure 3B), but significantly induced IL-5 and reduced TNFα plasma concentration ( Figure 3A). Although we observed no difference in systolic blood pressure between mice treated with sgp130Fc and the control mice ( Figure 3C), there was a significant reduction in aneurysm rupture after sgp130Fc treatment compared to control treatment ( Figure 3D).

Inhibition of IL-6 signalling pathway in elastase + anti-TGFβ mouse model
Using the elastase + anti-TGFβ model, we found that blockage of the IL-6R pathway using anti-IL-6R resulted in significantly increased mortality ( Figure 4A) induced by aortic rupture ( Figure   4B) but there was no change in the diameter of the aneurysm at the end of the experiment ( Figure   4C). αSMA + density in the media was similar in the two groups of mice (data not shown). There was also no change in the collagen content of the aortic wall ( Figure 4D) or the recruitment of myeloperoxidase positive (MPO + ) cells ( Figure 4E), but treatment with anti-IL-6R significantly enhanced the recruitment of CD3 + T cells in the aortic wall ( Figure 4F).
Blocking only the IL-6 trans-signalling pathway using sgp130Fc significantly increased survival ( Figure 5A) by reducing aortic ruptures ( Figure 5B), although at the end of the experiment there was no change in the aortic diameter between the treated and control mice ( Figure 5C). Histological analysis of aortic samples revealed a significant increase in the collagen content of the arterial wall ( Figure 5D) but no differences in αSMA + density (data not shown), Ly6G + ( Figure 5E) and CD3 + T cell accumulation ( Figure 5F) after sgp130Fc infusion, as compared to the control mice. Table 2 summarises the results of the different mouse models with a comparison to the human genetic data.

Evaluation of the effect of IL6R-rs2228145 on a range of cardiovascular markers
As would be expected, the minor allele of IL6R-rs2228145 was associated with increased plasma concentrations of IL-6 and sIL-6R. The variant was also associated with increased monocyte count, after correcting for multiple comparisons (p<1.389x10 -3 ). At a nominal significance level (p<0.05), rs2228145-C was associated with reduced lymphocyte count, increased levels of the cytokines CXCL10, CXCL11 and IL1α, as well as CD6 (an important regulator of T cells), and reduced levels of MMP3 (a matrix metalloproteinase), TIMP4 (a metalloproteinase inhibitor), OPG (osteoprotegerin) and IGFBP1 (Supplementary Figure 8). In our analysis, the effects on plasma IL-2, IL-5, and CXCL1 levels, as well as on blood pressure, were not statistically significant.

Discussion
In a combined analysis of the available worldwide clinical genetic data on AAA growth, we observed no statistically significant decrease in annual AAA growth rates for carriers of the minor allele of the Asp358Ala variant (rs2228145) in the IL6R gene. While we did observe a 15% decrease in the rate of reaching the surgery threshold of ≥55mm (HR=0.85 [0.73, 0.98] per copy of the minor allele), people with copies of the IL6R-Asp358Ala variant also had, on average, smaller baseline aneurysm diameters. Although we tried to account for this by allowing baseline hazards to vary depending on initial aneurysm size, some residual confounding is possible and could explain the observed results. In experimental data from mouse models, we found that selective blockade of the IL-6 trans-signalling pathway was associated with decreased aortic rupture and death. In exploratory analyses of cardiovascular and inflammatory biomarkers in healthy participants, we found that rs2228145-C was inversely associated with plasma levels of osteoprotegerin, matrix metalloproteinase-3 and metalloproteinase inhibitor-4 (p<0.05).
Osteoprotegerin has previously been shown to promote matrix metalloprotease release from monocytes and vascular smooth muscle cells, 31,32 and aberrant aortic extracellular matrix remodelling has been suggested to play a key role in the pathogenesis of AAA. 33 However, we note that further studies are needed to validate our biomarker data. Taken together, these human genetic, biomarker, and experimental murine findings are compatible with the concept that IL-6 trans-signalling is relevant to AAA growth, encouraging larger-scale evaluation of this hypothesis.
If increased availability of sIL-6R results in a dampening of the IL-6 trans-signalling pathway, this may explain potential protective effects in AAA and is consistent with previously observed protective effects in mouse models of sepsis 34 and pancreatic and lung failure. 35 As we found a consistent pattern of results when the trans-signalling pathway was selectively blocked in the mouse models, it suggests that this pathway could have a detrimental effect on AAA growth ( Figure 6). For example, the minor allele of the rs2228145 variant may result in a local reduction of IL-6 trans-signalling in the abdominal vasculature, reducing AAA risk 12 and, perhaps, AAA growth rates. Our observation of only a small, but statistically insignificant, decrease in annual AAA growth rates does not preclude meaningful clinical effects, since the growth rate reduction estimated from natural genetic variation does not necessarily relate to the magnitude of the benefit that might result from pharmacological treatment directed at the IL-6 trans-signalling pathway. 36 Selective blocking of the IL-6 trans-signalling pathway using sgp130Fc is being investigated in phase II clinical trials in patients with inflammatory bowel disease. 14 Nevertheless, apparent inconsistencies in our findings require further elucidation. For example, blockade of both the classic and trans-signalling IL-6 pathways using the animalequivalent (MR16-1) of tocilizumab had no effect on AAA rupture in the angiotensin II + anti-TGFβ model, but it was associated with decreased survival in the elastase + anti-TGFβ model.
These different outcomes could be explained by differences in the development of AAA in the different mouse models. The primary process of "aneurysm" formation in the angiotensin model is a medial dissection, which may be accentuated by elevations in blood pressure (even though high blood pressure is not the primary cause of medial dissection). Hence, the potential protection afforded by MR16-1 antibody in this model can at least in part be attributed to the significant reduction of blood pressure. In contrast, the elastase model does not involve medial dissection or elevations in blood pressure, but induces progressive remodelling, dilatation, and eventually transmural rupture of the artery wall, better mimicking AAA progression in humans. 37,38 If selective blockade of the IL-6 trans-signalling pathway results in decreased aortic rupture, as suggested by our murine data, one might expect that blocking both the classic and trans-signalling pathways would also result in decreased aortic rupture. However, we did not observe such a finding, perhaps due to competing downstream actions of the classic and transsignalling pathways. Such an explanation is consistent with our finding that IL-5 levels were Our study had potential limitations. It was powered to detect reductions in aneurysm growth of ~0.21mm per year or larger, much greater than the observed non-significant decrease of 0.06mm per year. Future studies powered to see an effect the same size as that observed in the current study would need to recruit an additional ~21,500 participants (total participants needed=24,444, Supplemental Material). This is unlikely to be achievable in the near future; alternative study methods using a composite phenotype for disease progression may be needed.
Index event and survival bias, in which participants are selected into the study based on both having and surviving an event, may have biased the results towards the null. 39 However, this bias is likely to be small (<10%). 39 Further, ultrasound, the primary method used to assess AAA diameter in the included studies, has a margin of error of 2-3mm, 40 greater than the annual rate of aneurysm growth, making changes in growth difficult to detect. This might be why we observed an association between the IL6R-Asp358Ala variant and time to surgery threshold of ≥55mm but not when looking at continuous change in AAA size. Although we examined rupture rather than aortic diameter as the outcome in the mouse experimental models, our published data indicate that aortas that rupture have larger diameters or faster diameter progression than the ones that do not rupture. 30 It is also uncertain how well the results of our animal models translate to clinical disease.
For example, an important difference is that IL-6 blockage is initiated before or at the time of disease development in the mice models of AAA, thereby not truly mimicking the treatment effects expected in humans, in which drugs to block IL-6 pathway would be started after disease onset. Blockade of the IL-6 signalling pathways in the angiotensin II + anti-TGFβ mouse model resulted in reproducible reductions in systolic blood pressure. Although tocilizumab has been anecdotally reported to improve pulmonary hypertension in Castleman's disease, 41, 42 the rs2228145 variant was not associated with changes in systemic blood pressure in healthy participants in a genome-wide association study. A large-scale randomized trial found no difference in the number of hypertension events reported in those using tocilizumab compared to placebo. 43,44 Thus, the acute responses to pharmacological doses of angiotensin II in the mouse model may not faithfully reproduce the human setting of AAA.
Our study may have clinical implications. Tocilizumab is currently indicated in a few disease settings, including rheumatoid arthritis and giant cell arteritis, both of which are associated with an increased risk of aortic aneurysm. 45,46 The development of coronary artery aneurysms has also been reported in a non-placebo-controlled pilot study of tocilizumab in children with Kawasaki's Disease. 47 Findings from this pilot study in children, combined with our finding that blocking the IL-6 pathway using the animal-equivalent of tocilizumab was associated with decreased survival in the elastase + anti-TGFβ model, suggests that patients treated with tocilizumab for conditions associated with aortic aneurysm development should possibly be monitored for AAA.
In conclusion, our proof-of-principle data are potentially compatible with the concept that IL-6 trans-signalling is relevant to AAA growth, encouraging larger-scale evaluation of this hypothesis.

Acknowledgments:
We thank Chugai Pharmaceutical Co., Ltd. for providing the MR16-1. We thank Tao Jiang and Praveen Surendran (Cardiovascular Epidemiology Unit, University of Cambridge) for providing access to genomic datasets.

Sources of Funding: The Cardiovascular Epidemiology Unit is supported by the UK Medical
Research Council (MR/L003120/1), British Heart Foundation (RG/13/13/30194), and National    ***p<0.05 isotype vs anti-IL6-R; 2-way ANOVA followed by uncorrected fisher's test. C -Systolic blood pressure measurement using tail cuff at day 0 and day 7 after angiotensin II and anti-TGFβ infusion. ***p<0.001 isotype vs anti-IL-6R; 2-way ANOVA followed by uncorrected fisher's test. D -Survival curves of mice after angiotensin II and anti-TGFβ infusion. All data for the generation of the graphs shown in Figure 2 were generated in two independent experiments and then pooled together.   Figure 4 were generated in one independent experiment.