Inflammatory innate lymphoid cells predict response speed to dupilumab in chronic rhinosinusitis with nasal polyps

Abstract

To the Editor, Chronic rhinosinusitis with nasal polyps (CRSwNP) is a condition characterized by chronic inflammation of the nose and paranasal sinuses, as well as the presence of bilateral nasal polyps (NP). A substantial fraction of CRSwNP patients remains uncontrolled, despite previous treatment with local and systemic corticosteroids, surgery, or both, which constitutes a significant socioeconomic burden.1 The pathological mechanisms underlying CRSwNP in Western societies are predominantly mediated by type 2 (T2) inflammation, including elevated levels of Interleukin- (IL)-4, IL-5, and IL-13, as well as infiltration of the NP tissue with eosinophils, mast cells, and group 2 innate lymphoid cells (ILC2s). ILC2s have emerged as a central regulator of T2 inflammation due to their capacity to produce copious amounts of T2 cytokines.2 Monoclonal antibodies targeting specific components of the T2 pathway are increasingly popular as a treatment option for CRSwNP. Dupilumab, a biological that blocks the IL-4/IL-13 alpha receptor, has been shown safe and effective in severe and uncontrolled CRSwNP patients.3 We report our findings from a real-life observational cohort, aiming to evaluate the predictive biomarkers for the response to dupilumab in n = 38 CRSwNP patients (≥18 years) in our tertiary referral center, as described before.4 We analyzed ILC populations with 18-parameter flow cytometry in the blood of eligible patients before the treatment was initiated (baseline) and the patients were categorized into three groups (fast-, normal-, and slow responders) based on the clinical outcomes at ≥12 weeks follow-up (full description in Data S1). Dupilumab was auto-administered subcutaneously, 300 mg 1 × /2 weeks (full methodology in Supplements). Our previous studies showed increased frequencies of ILC2s in the blood and NP tissues in CRSwNP; however, ILC2 frequency varied between individuals.5 We reasoned these variations may be linked to distinct differences between CRSwNP endotypes and may be reflected in the level of responses to dupilumab. Analyzing the expression of CRTH2 and NKp44, we observed increased frequencies of conventional ILC2 in the blood of patients responding fast to dupilumab and conversely increased frequencies of ILC3s in slow responders (Figure 1A–C). We have not found any differences across the patient groups for the expression of the hallmark transcription factors for ILC2 or ILC3—GATA-3 or RORχT, respectively (Figure S1A). Next to the conventional ILC2, we recently demonstrated increased frequencies of inflammatory CD45RO+ ILC2s in circulation and NP tissue of CRSwNP patients.6 CD45RO+ ILC2s are derived from resting CD45RA+ ILC2s upon exposure to activating, mucosal tissue-derived inflammatory cytokines. Upon the conversion, inflammatory ILC2s are resistant to corticosteroids and their frequencies correlate with disease severity.6 Here, we showed increased numbers of inflammatory ILC2s in fast responders, based on the increased expression of CD45RO and reduced CD62L (Figure 1A,D) as compared to normal and slow responders. Alongside, we found a trend toward increased frequencies of KLRG1+ ILCs in fast responders (Figure S1B), suggesting that the maturation status of ILCs varies across the patient groups. Further analysis of inflammatory ILC2s in fast responders revealed increased numbers of IL-13-producing CD45RO+ ILC2, but not IL-5+CD45RO+ ILC2s (Figure 1A,E). Given the mechanism of action of dupilumab, we evaluated the role of IL-4 and IL-13 in in vitro ILC2 activation and cytokine production. We found that IL-4 and IL-13 enhanced IL-13 production as compared to ILC2 cultured with TSLP plus IL-33 alone (Figure 2A) suggesting the role of dupilumab in modulating the ultimate IL-13 cytokine pool produced by ILC2s. Furthermore, the presence of IL-4, but not IL-13 potentiated IL-5 production levels (Figure 2B). In vitro stimulation with TSLP plus IL-33 of ILC2s pre-exposed for 48 h to dupilumab resulted in reduced IL-13 and IL-5 production by ILC2, as compared to TSLP plus IL-33 alone, while simultaneous exposure of ILC2s to TSLP plus IL-33 in the presence of dupilumab showed a trend toward the reduction of IL-13 and IL-5 (Figure 2C). Lastly, exposure of conventional CD45RA+ ILC2s to TSLP plus IL-33 in the presence of dupilumab did not prevent the conversion to the inflammatory CD45RO phenotype (Figure 2D), suggesting that the reduced production of type 2 cytokines is independent of the ILC2 activation and acquisition of the inflammatory phenotype. Limitations apply as well. Selection bias of the patients may have occurred due to this study's setting (tertiary referral center), possibly comprising the patients with the most severe and difficult-to-treat CRSwNP. Also, due to the small sample size of the study, results related to potential clinical relevance for patients' response prediction need to be interpreted with caution. Taken together, these data indicate the role of dupilumab in reducing the pool of IL-13 produced by ILC2s in CRSwNP and this reduction is likely directly linked to the improvement of patients' symptoms already at 4 weeks of treatment. The baseline frequencies of inflammatory CD45RO+ ILC2s may be a predictor for patients' responses to dupilumab and may potentially guide the choice of therapy in clinical practice. KG designed the study, did experiments, analyzed the data, and wrote the manuscript; RJLvdL provided samples and analyzed the data; DvE and EdG did experiments; HS, AMvZ, and CMvD analyzed the data; WJF and SR designed the study, analyzed the data, and wrote the manuscript. The authors would like to thank Y. te Winkel and I.M. Bruins for their assistance in patients' inclusions and study organization. KG was supported by funds from STIMAG and Amsterdam Institute for Infection and Immunity, Amsterdam UMC. RL has acted as a consultant and/or advisory board member for GSK. WF is an advisory board member of Sanofi, GSK, and Dianosic. SR has acted as a consultant and/or advisory board member for Sanofi, GSK, and Novartis. AMvZ has received research grants outside the submitted work from GSK, Boehringer Ingelheim, AbbVie and Vertex; she is the PI of P4O2 consortium, a public-private partnership co-funded by Health~Holland involving in-cash/in-kind contributors (Aparito, Boehringer Ingelheim, Breathomix, Clear, Danone Nutricia Research, Fluidda, MonitAir, Ncardia, Ortec Logiqcare, Philips, Quantib-U, RespiQ, Roche, Smartfish, SODAQ, Thirona, TopMD, and Novartis); she has served in advisory boards for AstraZeneca, GSK, and Boehringer Ingelheim. The Department of Otorhinolaryngology and Head/Neck Surgery of the Amsterdam UMC has received research funding from Sanofi, GSK, and Novartis. All the other authors declare that they have no conflict of interest. The data that support the findings of this study are available from the corresponding author upon reasonable request. Data S1. Figure S1. 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