Clinical C3 Inhibition With AMY-101 Reveals Novel Insights Into IL-8-Driven Inflammation in COVID-19

Complement overactivation drives the hyperinflammatory state of severe COVID-19, fueling a cycle of neutrophil-driven immunothrombosis, excessive cytokine release and endothelial injury [1-3]. In this context, neutrophils migrate to the lungs, releasing neutrophil extracellular traps (NETs) that interact with lung fibroblasts, which in turn amplify thromboinflammatory and immunofibrotic responses, further impacting disease progression [1, 4-7]. Interleukin-8 (IL-8) is a major neutrophil chemoattractant expressed by various cells, including neutrophils. Previous data have indicated that neutrophil-derived systemic and pulmonary IL-8 is overexpressed in COVID-19, aggravating disease immunopathology and prognosis [7, 8]. However, the role of complement in IL-8-associated disease pathology has yet to be clarified.

Considering and expanding upon the above findings, here we provide a novel mechanism that links complement C3 activation with IL-8-driven inflammation during neutrophil-lung fibroblast interaction in severe COVID-19, by leveraging clinical data and samples from the ITHACA study, the only randomised, placebo-controlled clinical trial of the compstatin-based C3 therapeutic AMY-101 in COVID-19-associated acute respiratory distress syndrome (ARDS) [9]. AMY-101 administration resulted in complete and sustained inhibition of C3 activation in all responders, alongside a reduction in thromboinflammatory markers associated with disease progression [9].

We analysed all 26 survivors of COVID-19-related ARDS, who had received either AMY-101 (5 mg/kg; n = 13) or placebo (n = 13), in addition to standard-of-care therapy, which included dexamethasone and low molecular weight heparin. To evaluate the in vivo effect of C3 inhibition on IL-8 expression, we measured IL-8 plasma levels in both study groups at baseline (D0) and at Day 7 post-treatment (D7). IL-8 plasma levels were found to be significantly reduced in the AMY-101-treated patients at D7 compared to D0. In contrast, this effect was not observed in the respective samples of placebo-treated patients (Figure 1A). Moreover, the key markers of complement activation, C3a and sC5b-9, including both D0 and D7 values, were found to be well correlated with the corresponding IL-8 levels (Figure 1B), suggesting a pathomechanistic role of complement activation in IL-8-associated inflammation during severe COVID-19.

Prompted by the above findings, we sought to investigate the potential cellular sources of IL-8 that may be modulated by C3 inhibition with AMY-101. Lung fibroblasts and neutrophils are considered key components of the maladaptive inflammatory response, leading to lung tissue damage and impaired respiratory function in COVID-19-associated ARDS [4, 6, 8, 10]. Incubation of healthy lung fibroblasts with D7 AMY-101 plasma yielded significantly lower IL-8 expression compared to the respective D0 samples (Figure 1C–F). This effect was not observed upon stimulation of lung fibroblasts with D7 placebo plasma (Figure 1C–F). Accordingly, the same pattern was observed in plasma-stimulated neutrophils (Supplementary Figure S1A–C). Taken together, these data indicate that the downregulation of IL-8 secretion from lung fibroblasts and neutrophils may be primarily attributed to C3 inhibition. Next, to address the potential functional implications of AMY-101's inhibitory effect on IL-8 secretion by lung fibroblasts, we measured neutrophil chemotactic activity using supernatants from COVID-19 plasma-stimulated lung fibroblasts. We found that supernatants obtained from lung fibroblasts activated with D7 AMY-101 plasma, exhibited diminished neutrophil chemotactic effect, compared to D0, employing IL-8 neutralisation to demonstrate that this response was IL-8-dependent (Figure 1G).

In addition to neutrophils [7], these results implicate lung fibroblasts as another source of increased IL-8 expression during severe COVID-19. Importantly, C3 inhibition with AMY-101 effectively abolishes this effect, uncovering C3-mediated IL-8-driven neutrophil recruitment into the lungs as a novel pathogenic mechanism in COVID-19-associated ARDS.

The effect of complement activation on lung fibroblasts is likely both direct and indirect. Lung fibroblasts express complement receptors, that is, C3aR and C5aR1 [11], allowing direct interaction with complement activation fragments, such as anaphylatoxins C3a and C5a, respectively. At the same time, complement activation in various disease models can induce pro-inflammatory cytokines, indirectly influencing fibroblasts by interacting with other cell types and modulating the inflammatory milieu [2]. Additionally, fibroblasts are well-established sources of IL-8 in response to other pro-inflammatory stimuli or pathogens [12, 13].

This study is the first to clinically validate the direct link between C3 activation and IL-8-driven inflammation, expanding our current understanding of COVID-19 pathophysiology and potentially other neutrophil-mediated thromboinflammatory and fibrotic disorders. In this context, targeting C3 may serve as an effective therapeutic strategy to mitigate multiple IL-8-related disease-exacerbating pathways [14, 15].

The experimental protocol regarding healthy neutrophils and lung fibroblasts was approved by the Scientific and Ethics Committees of the University Hospital of Alexandroupolis (Ref. No. 87/08-04-2020).

J.D.L. is the founder of Amyndas Pharmaceuticals, which is developing complement inhibitors (including third-generation compstatin analogues such as AMY-101). J.D.L. is an inventor of patents or patent applications that describe the use of complement inhibitors for therapeutic purposes, some of which are developed by Amyndas Pharmaceuticals. J.D.L. is also the inventor of the compstatin technology licenced to Apellis Pharmaceuticals (namely 4(1MeW)7W/POT-4/APL-1 and PEGylated derivatives such as APL-2/pegcetacoplan/Empaveli/Aspaveli/Syfovre). D.C.M. has provided consulting services to 4D Molecular Therapeutics, Rocket Pharma, Amyndas Pharmaceuticals and Merck KGaA. The other authors declare no conflicts of interest.