Introduction
Kawasaki disease (KD) is an acute systemic vasculitis of unknown etiology that affects small-sized and medium-sized arteries of infants and children. It is the main cause of acquired heart disease during childhood in developed countries. A single infusion of 2 g/kg of intravenous immunoglobulins (IVIG) along with aspirin has reduced the frequency of coronary artery aneurysms (CAA) from 25 to 5%. However, 10–20% of patients are unresponsive to IVIG treatment and thus present with persisting fever and inflammation, and have a threefold increased risk of cardiac complications and death [
1,
2].
Elevated levels of inflammatory cytokines such as tumor necrosis factor α (TNF) and IL-6 primed investigations of cytokine targeting strategies as rescue therapies in case of IVIG resistance. Yet, even though both therapeutic TNFα-neutralization [3] and IL-6R-blockade [
4] either in addition to IVIG treatment [3] or as prospective pilot study in IVIG-resistant cases [
4] contributed to improvement of some clinical and laboratory measures [
3,
4], these approaches did not reduce treatment resistance [
3]. Pilot study data regarding IL-6R-blockade even suggest an association of therapy with formation of new-onset CAA [
4].
Apart from TNF and IL-6, compelling evidence gathered over recent years points to a major role of NLRP3 inflammasome activation and IL-1 family cytokines in KD. This finding is all the more intriguing as it highlights a significant pathophysiological overlap between KD and systemic juvenile idiopathic arthritis (SJIA, Still’s disease), in addition to some of the common clinical features already noted, notably the potential progression to macrophage activation syndrome (MAS) [
5].
Historic data already demonstrated the prevalence of elevated IL-1 pathway gene signatures in KD [
6,
7]. Furthermore, spontaneous IL-1β release from in vitro cultured peripheral blood mononuclear cells (PBMCs) obtained from KD patients and responsive to IVIG treatment has been shown [
8]. Elevated circulating IL-1 and IL-18 serum levels separated acute KD children from febrile controls and decreased in course of convalescence [
9]. NLRP3-associated gene expression might reflect KD disease activity, while polymorphisms in genes encoding proteins involved in regulating intracellular Ca
2+ flux as a prominent NLRP3 inflammasome activator have been found to associate with KD [
9]. Inflammatory activation of human coronary artery endothelial cells showed to be particularly sensitive to IL-1β released from human monocytes upon toll-like receptor 4 stimulation and subsequent inflammasome activation [
10]. Similarly, disease development in the
Lactobacillus casei cell wall extract (LCWE)-induced KD mouse model required IL-1 producing macrophages as well as MyD88-signaling in hematopoietic cells [
11]. Mirroring an increased KD incidence in male patients, male mice subjected to the LCWE-induced KD mouse model also presented with a more severe IL-1β-dependent disease phenotype [
12]. Animals subjected to this pre-clinical KD model were protected from disease when receiving either IL-1α or IL-1β neutralizing antibodies, but protection was most evident when treatments were combined [
11,
13] or signaling through the IL-1 receptor was blocked using the recombinant IL-1 receptor antagonist (IL-1Ra) anakinra [
12,
14].
Built on this collective evidence, several clinical trials aiming to evaluate the efficacy of IL-1 blocking therapies in KD have been initiated [
15,
16]. Recently, results from the first open-label, phase IIa clinical trial (KAWAKINRA, Eudract Number: 2014–002,715-41, ClinicalTrials.gov NCT02390596) evaluating the safety of anakinra in IVIG-resistant KD patients have been published [
17]. These data support the early use of anakinra in IVIG-refractory cases, demonstrating that it is quickly effective on KD symptoms, inflammatory parameters, and coronary artery dilations in most patients, with good safety [
17]. Similarly, a second study assessing anakinra therapy in patients with acute KD and CAAs reported favorable safety and efficacy data [
16].
In this follow-up study, we investigated the impact of IL-1 blockade on circulating serum markers among KAWAKINRA participants and identified leucine-rich-α2-glycoprotein 1 (LRG1) to consistently associate with remnant inflammatory activity and the necessity to escalate anakinra dosage in IVIG-resistant KD patients and to separate those from sJIA-MAS as well as MIS-C. Both protein and retrospective analyses of historic gene expression data indicated tight association of LRG1 with IL-1β signaling and neutrophilia, while in vitro neutrophil stimulation with recombinant IL-1β resulted in concentration-dependent LRG1 release.
Discussion
In the investigated IVIG-resistant KD patients, inflammation upon study entry is hallmarked by over-expression of innate immune mediators, particularly IL-6, CXCL10, S100A12, and IL-1Ra. Subsequent treatment with anakinra decreased those most significantly, albeit almost all investigated blood biomarkers indicative of innate and/or adaptive immune mechanisms or (immune) cell activation indicated declining inflammation following IL-1R blockade. Throughout, increased serum LRG1 levels were associated with a subgroup of patients with elevated inflammatory activity, requiring escalation of anakinra dosage and separated those from (hyper)inflammatory conditions such as in sJIA-associated MAS or MIS-C. LRG1 expression on both protein and gene levels tightly associated with IL-1β levels and signaling as well as blood neutrophil counts, and IL-1 signaling in human neutrophils induced concentration-dependent LRG1 release.
Following IVIG unresponsiveness, we observed elevation of serum markers rather attributable to innate immunity, which reflects previous observations [
25]. Compared to healthy controls, particularly serum levels of IL-6 were strongly upregulated in our study cohort. IL-6 levels have been previously reported as among the most responsive to successful IVIG treatment [
26,
27], while persistent elevated levels can indicate refractory disease [
27]. Following anakinra treatment of IVIG non-responders, those as well as several others of the investigated marker levels normalized. This supports the proposed central role of IL-1 in KD pathophysiology and is in line with the previously reported clinical improvements observed in the study cohort upon anakinra treatment [
17].
Albeit approximately 80% of KAWAKINRA study patients became afebrile within 48 h following the last anakinra dose escalation and disease activity measures such as physician’s and parents’ evaluations as well as CRP levels improved in almost all cases [
17], in a subgroup of patients, serum levels of inflammatory markers and LRG1 in particular remained elevated throughout. Unbiased biomarker-driven hierarchical clustering analyses prior to and following 3 days of anakinra treatment suggested particularly elevated LRG1 concentrations to associate with persistent inflammation as reflected by respective clinical inflammatory parameters as well as the necessity to escalate anakinra dosage.
LRG1 behaves like an acute phase protein as it is mainly produced by hepatocytes following inflammatory stimulation. Beyond, it can be expressed by many cell types but particularly granulocytes, where it is suggested to have a role in cell differentiation [
22,
28]. By modulating TGF-β signaling, LRG1 is thought to be implicated in endothelial activation, vascular dysfunction, neovascularization, and cardiac re-modeling via fibroblast activation and cardiac fibrosis [
29,
30]. Importantly, genetic variation in the TGF-β pathway have been demonstrated to influence KD susceptibility, disease outcome, and response to therapy and are thought to support the importance of this pathway in KD pathogenesis [
31].
In context with KD, LRG1 has already been reported as elevated in sera from acute phase KD patients compared with individuals in the recovery phase and healthy controls [
32] and double positivity for both LRG1 and angiotensin has been suggested as biomarker for differentiating incomplete KD from non-KD patients [
33]. Furthermore, LRG1 has been found selectively upregulated in serum exosomes isolated from KD patients with CAAs [
34]. As also demonstrated by our data, this collectively suggests LRG1 expression and serum levels to reflect increased inflammatory activity in specifically KD.
Importantly, LRG1 expression on both protein and gene levels associated with IL-1β signaling as well as blood neutrophil counts, and IL-1β stimulation of particularly human neutrophils induced concentration-dependent LRG1 release. As already demonstrated elsewhere [
35], cells in whole blood remained comparably unresponsive to recombinant IL-1β, likely due to blockade or decoy of IL-1β by endogenous IL-1Ra and IL-1R2, respectively. Studies in context with other inflammatory conditions such as on adult-onset Still’s disease (AOSD) [
36] or Crohn’s disease [
37] similarly suggest an association of IL-1β and LRG1 expression. In contrast, studies linking LRG1 with IL-6 or TNF signaling report rather contradictory results [
36,
38,
39], which are thus partly in line [
37,
38] with the weak associations observed among our KD data.
Albeit LRG1 levels have been reported elevated in both KD and sJIA [
38], we observed selective over-expression of LRG1 in KD when compared to sJIA-associated MAS. Similar to MAS, LRG1 serum concentrations in MIS-C, which has been reported to share overlapping clinical features with KD [
23,
24], are not elevated compared to healthy controls and thus both sJIA-MAS and MIS-C clearly separate from at least a subpopulation of the investigated KD patients. Compared to KD, we observed less or no association of LRG1 with circulating IL-1β in both sJIA-MAS and MIS-C, which may point towards a different inflammatory pathophysiology with yet unclear involvement of IL-1 signaling [
23,
40].
While, collectively, our data may raise speculations for a previously undiscovered patho-mechanistic involvement of an IL-1β-LRG1 axis as driver of cardiac re-modeling in KD, it still requires appropriate models and approaches to test for this. In a substantially larger patient cohort, we are now validating whether LRG1 serum levels can indeed inform on KD disease course and/or differentiate KD and MIS-C. Similarly, therapeutic targeting of LRG1 is being tested. In other disease context, therapeutic LRG1 inhibition has already been suggested to restore normal vascular function [
41,
42]. In the light of recent findings on depletion of bioactive IL-1β-loaded neutrophils as a central therapeutic mechanism in KD [
43], one may further speculate that in IVIG-resistant KD particularly those cells may escape this depletion, which may be supported by our data on the prevalence of an IL-1β-LRG1-neutrophil axis in the present study population. In the biomarker context of the present study, IL-6, S100A12, and LRG1 could offer some predictive power on the response of IVIG-resistant KD patients to therapeutic IL-1 blockade; however, data on the predictive potential of these markers already prior to start of anakinra therapy were inconclusive due to lack of statistical power within the small study population. Nevertheless, conventional markers of inflammation such as peripheral leukocyte and neutrophil counts as well as CRP levels were indeed able to indicate a future anakinra dose adjustment despite the small cohort size.
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