Discussion and Conclusions
The results of this study demonstrate that targeting the IL-5 pathway
with mAbs is a potent and effective strategy to prevent in a
concentration dependent manner the AHR in passively sensitized airways,
a condition leading to a massive production of IL-5 in bronchial tissue.
Benralizumab and mepolizumab reached the primary endpoint since both the
agents flattened and shifted rightward the CRC to histamine. Lower
concentrations of benralizumab than mepolizumab significantly reduced
the AHR to histamine, with benralizumab being also most effective than
mepolizumab by reducing the myogenic response at lower levels than that
detected in non-hyperresponsive bronchi. The advantage of blocking
IL-5Rα to IL-5 was further confirmed by the analysis of the potency,
since benralizumab was ≃0.7 logarithm more potent than mepolizumab in
reducing the contractile plateau generated by histamine in
hyperresponsive airways.
Concerning the secondary endpoints, although the efficacy in preventing
the AHR to parasympathetic stimulation was similar between the
investigated mAbs, benralizumab was more potent than mepolizumab when
the tissues were stimulated by higher EFS frequencies. Furthermore,
lower concentrations of benralizumab than mepolizumab were sufficient to
significantly counteract the FRC to EFS in hyperresponsive bronchi.
Benralizumab and mepolizumab also prevented the hyperresponsive myogenic
tone in response to QS, although only benralizumab reduced the ASM
contractility at a level lower than that detectable in non-sensitized
airways. Again, at lower concentrations (10 μg/ml) benralizumab but not
mepolizumab was effective in inhibiting the AHR to QS.
As previously reported (Cazzola et al., 2016b), also in this study the
passive sensitization altered the concentration of cAMP in
hyperresponsive airways, a condition that was reverted in a
concentration dependent manner by both benralizumab and mepolizumab.
Benralizumab, but not mepolizumab, improved the cAMP concentrations at a
level greater that that detected in non-sensitized tissue.
Certainly several complex indirect mechanisms (Molfino et al., 2012;
Mukherjee et al., 2014) are involved in the prevention of AHR elicited
by the inhibition of the IL-5/IL-5Rα axis in hyperresponsive airways.
Whatever the pathways implicated, we have found that benralizumab and
mepolizumab can restore at physiological levels the cAMP concentrations
in passively sensitized airways. Increased intracellular concentrations
of cAMP in ASM promote bronchodilation via activation of cAMP-dependent
protein kinase (A kinase) (Rogliani et al., 2016). However, conflicting
findings are currently available regarding the real impact of cAMP on
airway inflammation, with some evidences suggesting for
anti-inflammatory effects and other reporting increased cytokine
synthesis and T cell adhesion to ASM cells induced by enhanced
concentrations of cAMP (Black et al., 2012). Isolated airways include
several residential cells such as ASM cells, fibroblasts,
parasympathetic cells, epithelial cells, and inflammatory cells such as
T-helper type 2 (Th2) lymphocytes, eosinophils, and mast cells, most of
them stimulated or activated in response to passive sensitization
(Schmidt et al., 2000). Therefore, in the isolated airways used in our
experiments it was not possible to discern the exact origin of cAMP
enhancement induced by the investigated mAbs. However, the strong
correlation between the cAMP levels and the inhibition of AHR
contractility induced by benralizumab and mepolizumab suggests that the
improvement in cAMP concentrations represents a key mechanism on which
the inhibition of IL-5/IL-5Rα axis may converge and, thus, protect ASM
from hyperresponsiveness.
Unexpectedly, in passively sensitized airways benralizumab but not
mepolizumab improved AHR and cAMP at levels greater than those detected
in untreated non-sensitized tissue. Isolated airways are characterized
by a certain degree of intrinsic tone mediated by the spontaneous
generation of histamine and leukotrienes. Therefore, since eosinophil
secretory granules contain both histaminase and leukotrienes (Bagnasco
et al., 2017; Bandeira-Melo et al., 2002), it was expected that
preventing the activation and degranulation of eosinophils by
administering either benralizumab or mepolizumab would have led to the
same effect. Perhaps, the difference in the efficacy between
benralizumab than mepolizumab can be explained by considering that
although both these mAbs counteract the IL-5 pathway, only benralizumab
induces ADCC leading to the apoptosis of eosinophils and mast cells
caused by natural killer (NK) cells (Kolbeck et al., 2010). Thus, the
combination of IL5Rα blockade and ADCC activity of benralizumab may also
support the greater potency of this mAb compared to mepolizumab against
the AHR to histamine. Furthermore, while at low concentrations
benralizumab can antagonize IL-5Rα and elicit ADCC (Kolbeck et al.,
2010), low concentrations of mepolizumab could not be sufficient to
neutralize the massive release of IL-5 in the sensitized tissue.
Such a condition can be translated to asthmatic patients before the next
dose administration of a mAb. Accordingly with pharmacokinetic (PK)
studies in healthy subjects (Martin et al., 2019; Shabbir et al., 2019)
that received approved doses of mAbs (European Medicines Agency, 2015;
European Medicines Agency, 2018; US Food and Drug Administration, 2015;
US Food and Drug Administration, 2017), while the maximum plasma
concentrations of benralizumab and mepolizumab were ≃3 μg/ml and ≃12
μg/ml respectively, the trough concentrations were ≃1 μg/ml and ≃5 μg/ml
respectively. Indeed, our results indicate that at these concentrations
both the agents are effective in submaximally inhibit the AHR to
histamine in isolated airways. However, while the efficacy of the
circulating levels of mepolizumab at trough could be neutralized by the
cytokine storm during an asthma exacerbation (Borish, 2016), the
protective effect of low concentrations of benralizumab can be preserved
as it is directed on IL-5Rα and ADCC regardless of the the amount of
circulating IL-5.
Taken together the data from PK studies with those of our study, it is
also evident that the concentrations of benralizumab and mepolizumab
detectable in plasma can only partially prevent the AHR induced by
parasympathetic activation. This translational evidence is of certain
interest to optimize the pharmacological treatment of severe asthmatics
in which a high intrinsic parasympathetic tone has been documented
(Liccardi et al., 2016). Probably the current Global Initiative for
asthma (GINA) recommendations (GINA, 2019) should be improved by
considering this specific asthma phenotype in Step 5, in which combining
an anti-IL-5/IL-5Rα mAb with a long-acting muscarinic antagonist (LAMA)
as preferred controller could lead to clinical and functional benefits.
Airway sensitization contributes to the adhesion of eosinophils to
parasympathetic nerves, leading to their priming, activation and
degranulation with consequent release of major basic protein (MBP). MBP
increases acetylcholine release due to loss of function of the neuronal
M2 muscarinic autoreceptor expressed on postganglionic
parasympathetic neurons (Drake et al., 2018). Since such an intimate
interaction between eosinophils and airway cholinergic nerves
contributes to the AHR in the course of tissue sensitization, favourable
synergistic interaction could result by combining a LAMA, that inhibits
the cholinergic transmission, with an anti-IL-5/IL-5Rα mAb that protects
vagal fibres from the deleterious influence of activated eosinophils.
By a strict pharmacological viewpoint, the findings of our study suggest
that the profile of loss of Emax to histamine and EFS in
passively sensitized airways treated with either benralizumab and
mepolizumab is due to an indirect inhibition of endogenous
bronchoconstricting intermediaries release that, in turn, lead to the
AHR. This evidence is supported by the fact that the AHR induced by
histamine in passively sensitized bronchi is mediated not only by the
direct activation of histaminergic receptors expressed on ASM, but also
by the indirect facilitator effect of the acetylcholine release from the
parasympathetic nerve (Cazzola et al., 2016a). Moreover, the AHR
elicited by EFS is prevalently indirect and mediated by the
sensitization of vagal fibres leading to increased release of endogenous
acetylcholine from postganglionic parasympathetic nerves that in turn
activate muscarinic M3 receptor expressed on ASM
(Ichinose et al., 1996; Mitchell et al., 1993).
This research provides also ancillary results, mainly concerning the
validity of passive sensitization of human isolated airways as a
suitable model of AHR in asthma. Although already demonstrated at human
ASM cellular level (Grunstein et al., 2002; Hakonarson et al., 1999a;
Hakonarson et al., 1999b), here we provide for the first time the
evidence that passive sensitization of the whole human bronchial tissue
induces an extensive synthesis of IL-5, and that IL-5 represents a key
factor leading to the AHR in human airways. Certainly targeting the IL-5
pathway counteracts the AHR in human subsegmental bronchi, nevertheless
Manson et al. (Manson et al., 2019) have recently demonstrated that IL-5
does not induces hyperresponsiveness in human small airways. Perhaps the
lack of IL-5 mediated AHR in small airways could be due to the
preponderance of ASM in the bronchioles wall and the significantly
lower, and almost absent, number of eosinophils in bronchioles compared
to medium bronchi we have used in our experiments (Faul et al., 1997;
Hyde et al., 2009). Furthermore, a possible bias leading to the absence
of AHR in the bronchioles used by Manson et al. (Manson et al., 2019) is
that their tissues were not passively sensitized, a procedure that
induces AHR via IgE in the presence of further serum factors (Ichinose
et al., 1996; Mitchell et al., 1997; Schmidt et al., 2000; Schmidt et
al., 1999). In our study we have also found that the passive
sensitization of subsegmental bronchi (4-6 mm inner diameter) elicits
AHR in response to EFS. Since no augmentation to EFS was detected in
previous studies in smaller airways (2-3 mm inner diameter) regardless
of the frequency delivered (Mitchell et al., 1997), it can be assumed
that the distribution of functional parasympathetic innervation at the
level of airways ≤3 mm is sparse and/or hyporesponsive to passive
sensitization (Calzetta et al., 2018a), making these smaller airways not
appropriate to reproduce ex vivo the AHR mediated by vagal activation.
The main limitation of this study is intrinsic to the isolated bronchial
model itself, as it permitted to characterize the effect of benralizumab
and mepolizumab against AHR in a sub-acute ex vivo experimental setting
but not after a long-term exposure. In this regard, since AHR has been
proposed to be a main treatable trait toward precision medicine in
eosinophilic asthmatic patients (Agusti et al., 2016; Bel et al., 2017),
well designed head-to-head clinical trials are needed to compare the
efficacy of chronic treatment with benralizumab and mepolizumab
specifically against AHR in severe asthma.
Taken together, the findings of this study demonstrate that passive
sensitization induces a massive release of IL-5 from human airways, and
that targeting the IL-5/IL-5Rα axis with mAbs prevent in a concentration
dependent manner the AHR in response to histamine, parasympathetic
activation, and mechanical stress. Benralizumab, by blocking the IL-5Rα,
resulted more potent than the anti-IL-5 mepolizumab, and the beneficial
effects of both these agents were correlated with improved levels of
cAMP in hyperresponsive airways. These observations also indicate that
IL-5 is a key factor in determining AHR in passively sensitized airways.
Further head-to-head studies evaluating the impact of long term
treatment with benralizumab and mepolizimab against AHR are needed in
severe asthmatic subjects, with specific focus on patients characterized
by a high intrinsic parasympathetic tone.