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Basophils prime group 2 innate lymphoid cells for neuropeptide-mediated inhibition

Type 2 cytokine responses promote parasitic immunity and initiate tissue repair but, can also result in immunopathologies when not properly restricted. Basophilia is recognized as a common feature of type 2 inflammation, however, the roles basophils play in regulating these responses remain unknown. Here, we demonstrate that helminth-induced ILC2 responses are exaggerated in the absence of basophils, resulting in increased inflammation and diminished lung function. Additionally, we show that ILC2s from basophil-depleted mice express reduced amounts of the receptor for the neuropeptide, neuromedin B (NMB). Critically, NMB stimulation inhibited ILC2 responses from control but not basophil-depleted mice, and basophils were sufficient to directly enhance NMB receptor (NMBR) expression on ILC2s. These studies suggest that basophils prime ILC2s to respond to neuron-derived signals necessary to maintain tissue integrity. Further, these data provide mechanistic insight into the functions of basophils and identify NMB as a potent inhibitor of type 2 inflammation.


Introduction:
The World Health Organization estimates that helminth infections affect approximately 2 billion people worldwide, causing malnutrition, growth impairment, cognitive deficiencies, and chronic immunopathology 1,2 . Although antihelminthic drugs are available, they have short-lived effects, resulting in high reinfection rates within months of treatment 3 . Further, the generation of long-lasting immunotherapeutics has been precluded by our incomplete understanding of the mechanisms that properly regulate type 2 cytokine responses and host protection 4 . Therefore, developing a better understanding of the immunologic mechanisms that promote and/or inhibit type 2 inflammation would greatly inform the generation of new strategies to treat helminth infections and their associated morbidities.
Many helminths, such as hookworms, undertake complex migratory cycles that require their passage through multiple mucosal sites, including the lungs and gut. The passage of these large multicellular parasites is known to result in substantial wounding and tissue damage 5,6 . In response to these signals, it is well-established that the mammalian host mounts potent type 2 cytokine responses, characterized by the secretion of interleukin (IL)-4, IL-5, and IL-13, which simultaneously promote parasite expulsion and the healing of affected tissues 7 . When properly regulated, type 2 cytokine responses are limited in order to reduce the detrimental effects of prolonged tissue remodeling and fibrosis 8 . The importance of balancing both resistance and tolerance mechanisms is evident by the severe chronic immunopathologies that can result if anti-helminth immunity is not properly regulated 8,9 .
Elevated numbers of basophils in the periphery is a highly conserved characteristic of type 2 cytokine responses 10,11 . Although basophils are known to secrete robust amounts of IL-4 upon activation, basophils are also capable of producing several soluble mediators including cytokines (IL-6, TNF, and IL-13), inflammatory molecules (histamine and platelet-activating factor), bioactive lipids (prostaglandins and leukotrienes), as well as growth factors (amphiregulin and CSF-1) 10,11 . Given their capacity to secrete these potent effector molecules, it is not surprising that basophils are reported to operate as important mediators of allergic inflammation [12][13][14] . However, recent work has also begun to highlight the critical contributions of basophils during homeostatic conditions. For example, recent studies have demonstrated that basophils imprint on alveolar macrophages and play important roles during lung development, suggesting that the functions of basophils are context-dependent and extend beyond their ability to promote inflammation 15,16 .
Consistent with their functional diversity in response to distinct stimuli, the roles basophils play in the context of antihelminth immunity appear to be parasite-specific. While studies have demonstrated that basophils promote type 2 inflammation and worm expulsion following Trichuris muris and Trichinella spiralis infections, their functions following other helminth infections remain unknown [17][18][19] . Although the population expansion of basophils in response to the hookworm Nippostrongylus brasiliensis (Nb) has been appreciated for several decades, the functions of Nb-induced basophils remain poorly understood. Further, important studies have demonstrated that depleting basophils following a primary Nb infection has no effect on worm clearance 20,21 . These data suggest that Nb-induced basophils perform important functions that have yet to be appreciated.
A growing body of evidence has demonstrated that specialized innate immune cells engage in cellular cross-talk and cooperate to initiate type 2 inflammation 6,22 . For example, basophils can promote the activation of group 2 innate lymphoid cells (ILC2s) via their secretion of IL-4 12,14 . Additionally, recent reports show that neuron-derived signals such as neuromedin U (NMU) and calcitonin gene-related peptide (CGRP) are also capable of regulating the activation of ILC2s and thereby dictating the intensity of type 2 inflammation [23][24][25][26][27] . Collectively, these studies suggest that intra-communication (immune cell to immune cell) and extra-communication (immune cell to neuron) might cooperate to direct the scope of type 2 responses. Despite these advances, whether immune cell-cross talk is required to facilitate subsequent neuroimmune interactions remains to be defined.
Here, we report that Nb-induced basophils accumulate in the lungs following the exit of parasitic larvae from the tissue, suggesting that they might contribute to host protection by promoting the healing of helminth-affected tissues rather than directly acting on the parasites. Consistent with this hypothesis, our data demonstrate that Nb-induced ILC2 responses are exaggerated in the absence of basophils, resulting in increased inflammation and reduced pulmonary function. Critically, overactive ILC2s from basophil-depleted mice exhibited reduced expression of the receptor for neuromedin B (NMB), a member of the neuromedin family of neuropeptides that include neuromedin B, C, K, L, N, U, and S 28,29 . Importantly, treatment with exogenous NMB was sufficient to significantly reduce ILC2 responses, eosinophilia, and mucus production following Nb infection. Finally, while recombinant (r)NMB treatment inhibited sort-purified ILC2s from control mice, ILC2s from basophil-depleted mice were unresponsive to NMB stimulation, suggesting that basophils prime ILC2s for NMB-mediated inhibition. Altogether, our data highlight a previously unappreciated regulatory role for basophils in the context of antihelminth immunity and identify NMB as a potent inhibitor of type 2 inflammation.

Results:
Depletion of basophils results in severe helminth-induced pathology and impaired lung function.
Despite basophilia being a hallmark of type 2 inflammation 10,11 , the roles these enigmatic cells play in regulating type 2 cytokine responses remain controversial 20,21 . Therefore, we sought to better determine the functions of these rare immune cells following infection with the gastrointestinal nematode, Nippostrongylus brasiliensis (Nb). First, we evaluated several parameters of type 2 cytokine-mediated inflammation induced by Nb in the presence or absence of basophils. Lineage-specific depletion of basophils was achieved by administration of diphtheria toxin to Mcpt8 tm1(cre)Lksy ROSA26iDTR mice, as previously described 21 . While depletion did not alter inflammation in the gastrointestinal tract (Extended Data Fig. 1a) or worm expulsion (Fig. 1a), basophil depletion resulted in significantly increased type 2 cytokine responses in the lung (Fig. 1b), where the parasites transiently reside as part of their developmental life cycle. In vivo staining protocols used to distinguish blood-circulating versus tissue-resident cells revealed that tissue-resident basophil populations increased in the lungs starting on day 3 and peaked on day 5 post-infection (Fig. 1c, d). While effector cells activated in the context of type 2 cytokine responses are required to promote parasite clearance, they are also important for maintaining the integrity of parasite-affected tissues 7,22 . Since Nb larvae exit the lung tissue on day 3 post-infection, before the majority of basophils are present, it is possible that basophils are playing a role in regulating inflammation in an attempt to restore lung function rather than serving to limit parasitic burdens. In support of this hypothesis, Nb-infected mice depleted of basophils exhibited altered lung pathology, marked by increased mucus production (Extended Data Fig. 1b, c), inflammatory cell infiltrates (Fig. 1e, Extended Data Fig. 1d) and had significantly reduced oxygen levels compared to control mice (Fig. 1f). Collectively, these data provoke the hypothesis that basophils limit infection-induced type 2 cytokine responses and assist in maintaining lung function post-Nb infection.

Basophil depletion results in elevated ILC2 responses
Microscopic analysis of lung pathology suggested that basophil-depleted mice had elevated infection-induced eosinophil responses. Flow cytometric analysis of bronchoalveolar lavage (BAL) fluid and lung infiltrates confirmed that while infection-induced neutrophilia was not significantly altered ( Fig. 2a), (Extended Data Fig. 2a), basophil-depleted mice exhibited significantly increased BAL and lung eosinophil responses (Fig. 2b), (Extended Data Fig.  2b), as defined by flow cytometric gating strategies (Extended Data Fig. 2c). Nb-induced eosinophil responses and mucus production are dependent on IL-5 and IL-13, which are produced by ILC2s or CD4 + T cells 6,7 . Lung basophil responses occur in the first few days post-infection (day 3-5), during the innate window when Nb-induced inflammation is reported to be ILC2-dependent 6,22 . Further, the ability of basophils to communicate with and alter the activation state of ILC2s has been previously demonstrated 12,14 . Therefore, we sought to determine if basophil-depleted animals exhibited elevated infection-induced ILC2 responses that correlated with increased mucus production and eosinophilia. Interestingly, ILC2 populations were increased in both the BAL and lung tissue of basophil-depleted mice compared to controls (Fig. 2c), (Extended Data Fig. 2d). Further, increases in IL-5 and IL-13 producing ILC2s were also detected (Fig. 2d, e), (Extended Data Fig. 2e, f), as defined by specific gating strategies (Extended Data Fig. 2g). Similar results were also observed in the BAL and lungs of infected Mcpt8Cre mice that constitutively lack basophils 20 (Extended Data Fig. 3a-f). However, despite increased IL-5 and IL-13 production by lung ILC2s, no increases in infection-induced eosinophil responses were observed in the lungs of Mcpt8Cre mice (Extended Data Fig. 3f). This is likely due to compensation mechanisms that are thought to exist in this constitutive mouse model 11 . Importantly, basophil depletion in naïve mice did not alter ILC2 responses, suggesting that the observed differences are not due to diphtheria toxin receptor (DTR)-mediated cell death ( Fig. 2c-e, Extended Data Fig. 2d-f). Nonetheless, to further rule out the possibility of off-target depletion effects, we next took a gain-of-function approach and transferred DTR negative basophils into basophil-depleted mice. Critically, the intratracheal transfer of DTR negative basophils was sufficient to suppress ILC2 responses, mucus production, and eosinophilia back to normal levels in both the BAL and lungs of basophil-depleted mice ( Fig. 2f-i), (Extended Data Fig. 4a-g). Collectively, these loss-and gain-of-function approaches suggest that basophils negatively regulate lung ILC2 responses following a Nb infection.

Helminth-activated ILC2s exhibit reduced neuromedin B receptor expression in the absence of basophils
To further examine whether the effects of basophil depletion were occurring independently of adaptive lymphocytes, recombination-activating gene 2 (Rag2)-deficient mice were treated with the basophil-depleting antibody Ba103 30 . Rag2 −/− mice treated with Ba103 exhibited significantly elevated Nb-induced ILC2 responses and eosinophilia in both the BAL (Fig. 3a, b) and lung (Fig. 3c, d). Further, Rag2 −/− mice treated with Ba103 also exhibited significantly elevated mucus production and altered lung pathology (Fig. 3e-f). Similar results were observed when basophils were depleted by treatment with the FcεRIαspecific antibody MAR-1 19 (Extended Data Fig. 5a-f). Collectively, these data suggest that basophils negatively regulate lung ILC2 responses independently of adaptive lymphocytes. To elucidate the mechanism through which basophils regulate ILC2s, we first examined whether basophil depletion resulted in increased production of cytokine alarmins such as IL-25, IL-33, and TSLP, which promote ILC2 activation 31 . Importantly, no changes in the expression of Il25 or Tslp were observed in the lungs of basophil-depleted mice. However, basophil-depleted mice exhibited significantly reduced expression of Il33 in whole lung tissue (Extended Data Fig. 6a-c). Consistent with these data, analysis of IL-33-GFP reporter mice revealed that type 1 and type 2 pneumocytes expressed significantly less Nb-induced Il33 following treatment with the anti-FcεRIα targeting antibody MAR-1 (Extended Data Fig. 6d, e). In aggregate, these studies suggest that elevated ILC2 responses in the basophil-depleted mice are not a result of increased cytokine/alarmin production. We next evaluated whether basophils suppress ILC2 activation via regulating mediators including anti-inflammatory cytokines, such as IL-10, or growth factors, like amphiregulin. Although no significant changes in Il10 expression were detected in basophil-depleted mice, expression of amphiregulin was found to be significantly elevated in the absence of basophils (Extended Data Fig. 6f, g). Further, while basophils secreted robust amounts of activation-induced IL-6, we were unable to find detectable levels of basophil-derived IL-10 or amphiregulin (Extended Data Fig. 6h-j). Collectively, these data suggest that basophils are not regulating lung inflammation by producing these regulatory factors or amplifying their expression levels. Next, we performed single-cell RNA sequencing analysis to evaluate if the depletion of basophils substantially altered the composition of ILC2 populations. Single-cell RNA sequencing of lung-resident ILC2s revealed the presence of 6 distinct cell clusters defined by specific markers genes using the Seurat workflow (Extended Data Fig.  7a, b). Importantly, while basophil depletion did not result in substantial changes to the composition of ILC2s, helminth-induced expression of Il5, Il13, and Areg were significantly elevated across clusters of ILC2s sort-purified from basophil-depleted mice compared to control samples (Extended Data Fig. 7c). However, we did not detect significant changes in the expression of Il1rl1 and Arg1.
While single-cell RNA sequencing is helpful to identify general differences across samples, the depth of gene lists can make it difficult to perform pathways analysis 32 . To address this, we also performed conventional RNA-sequencing of lung ILC2s post-Nb, to better identify how basophils may regulate their state of activation. Control ILC2s expressed a substantial number of genes at significantly higher levels than those from basophil-depleted mice (Fig. 4a, Extended Data Fig. 8a), (Supplementary table 1). Interestingly, pathway analysis revealed that ILC2s from control animals were enriched for pathways including neuroactive ligand-receptor interactions, seven-pass-transmembrane domain receptor (7TM), G-proteincoupled receptor signaling, and rhodopsin-like signaling compared to ILC2s sort-purified from basophil-depleted mice (Fig. 4b). Notably, all three of these pathways are associated with neurotransmitter signaling, which has recently been identified as an important component of ILC2 activation post-Nb infection [23][24][25][26][27] . Specifically, the neuropeptide, neuromedin U (NMU) produced by cholinergic neurons promotes ILC2 activation post-Nb 23,24 . Importantly, NMU is part of the neuromedin family of neuropeptides including B, C, K, L, N, S, and U 29 . Similar to NMU, neuromedin B (NMB) is expressed in the central nervous system, lungs, gastrointestinal tract, and adipose tissues of mammals 28,29 . Further, both NMU and NMB signal through receptors belonging to the rhodopsin-like subfamily A7 of G-coupled protein receptors 28,29 , which was identified by pathway analysis. While no significant changes in the receptors for NMU (NMUR1) or NMB (NMBR) were identified by RNA-sequencing analysis on day 5 post-infection, when ILC2s were sort-purified from the lungs of mice on day 7 post-Nb (following the peak of lung basophilia), significant reductions in Nmbr expression were observed in basophil-deficient animals (Fig. 4c).
Further, while surface expression of NMBR by ILC2s was increased post-infection, ILC2s from basophil-depleted mice exhibited significantly reduced surface levels of NMBR protein (Fig. 4d, e). Interestingly, NMBR expression in alveolar macrophages was not altered when basophils were depleted (Fig. 4f), suggesting that basophils regulate NMBR expression in a cell-specific manner. Notably, expression of Nmu and Nmb ligand in the lung remained unchanged following basophil depletion (Fig. 4g). Collectively, these data provoke the hypothesis that basophils regulate ILC2 responses by altering their ability to respond to NMB-mediated signals.

Neuromedin B is a potent inhibitor of Nb-induced type 2 cytokine responses
To investigate if NMB operates as a negative regulator of type 2 cytokine responses, wild-type mice were infected with Nb and treated with rNMB. On day 7 post-infection, parameters of type 2 cytokine-dependent inflammation were evaluated in both the BAL and lung. Strikingly, rNMB-treated wild-type mice exhibited reduced ILC2 responses and eosinophilia in both compartments ( Fig. 5a-f). Further, rNMB treated mice also showed increased disruption of the alveoli, significantly reduced mucus production, and failed to clear worms as efficiently as control mice ( Fig. 5g-j). Collectively, this gain-of-function approach further suggests that NMB operates as a potent inhibitor of type 2 inflammation.
While NMB is reported to regulate cell growth, body temperature, blood pressure, and glucose levels via its effects on non-hematopoietic cells, its ability to alter the activation state of immune cells remains unknown 29 . To address this, we first evaluated expression of NMBR by CD45 + immune cells and found that NMBR is expressed by ILC2s, CD4 + T cells, alveolar macrophages, non-alveolar macrophages and eosinophils (Extended Data Fig.  8b-f). Collectively, these data suggest that NMB may influence Nb-induced inflammation via its effects on several cells of hematopoietic origin. To further evaluate whether NMB-NMBR signaling is important in the hematopoietic compartment, we generated NMBRfloxed mice (Extended Data Fig. 9a) and crossed them with Vav-iCre mice to remove NMBR expression from CD45-expressing cells 33 . As expected, NMBR was significantly reduced on CD45 + cells, including ILC2s, but remained unchanged in the non-hematopoietic compartment ( Fig. 6a-c). Similar to basophil depletion, NMBR fl/fl x Vav-iCre + mice exhibited significantly increased expression of Il4, Il5 and Il13 in lungs on day 7 post-Nb infection (Extended Data Fig. 9b-d). Critically, NMBR fl/fl x Vav-iCre + mice also presented with significantly elevated cytokine responses by ILC2s and elevated eosinophilia in both the BAL (Fig. 6d-h) and lungs (Extended Data Fig. 9e-g) post-Nb. Additionally, selectively targeting NMBR expression in the hematopoietic compartment also resulted in significantly elevated Muc5ac expression and increased cellular infiltrates in the lung (Fig. 6i, j).
Collectively, these data suggest that NMB-NMBR signaling on immune cells is required to properly regulate Nb-induced inflammation.

Neuromedin B is a potent inhibitor of innate immunity to Nb
The data above suggest that basophils regulate NMB-NMBR-signaling that is required to limit type 2 cytokine responses post-Nb. Further, these studies suggest that NMB may act on cells in both the innate and adaptive immune compartments. Nb-induced basophils enter the lungs between days 3-7 post-infection ( Fig. 1d) when type 2 cytokine responses are reported to be primarily ILC2 driven 6,22 . Therefore, we sought to determine if NMB can negatively regulate Nb-induced inflammation independently of adaptive lymphocytes. To test this, Rag2 −/− mice were infected with Nb and treated with rNMB. Consistent with experiments in wild-type mice, Rag2 −/− mice treated with rNMB exhibited significantly reduced ILC2 and eosinophil responses in both the BAL and lung (Fig. 7a-c), (Extended Data Fig. 9h-k). Further, rNMB-treated Rag2 −/− mice also exhibited significantly reduced mucus production and impaired worm expulsion, suggesting that rNMB treatment is sufficient to inhibit type 2 inflammation in the absence of adaptive lymphocytes (Fig. 7d, e). rNMB-treated Rag2 −/− mice also presented with significantly elevated expression of Il17 and increased neutrophilia, suggesting that in the absence of type 2 cytokine production, early Nb-induced IL-17 responses remain elevated 34,35 (Fig.7f-g, Extended Data Fig. 9l). Finally, rNMB treatment resulted in severe lung pathology and significantly elevated numbers of red blood cells in the BAL, an indication of a reduced capacity to initiate type 2-dependent wound healing 36,37 ( Fig. 7h-j). Collectively, these data suggest that NMB operates as a potent negative regulator of innate type 2 responses.

Neuromedin B directly inhibits ILC2s
The above studies demonstrate that NMBR is expressed by several innate cell populations known to promote anti-helminth immunity. Further, gain-of-function studies demonstrate that rNMB is sufficient to inhibit Nb-induced ILCs responses in vivo. However, whether NMB acts directly on ILC2s remains unknown. To test this, we sort-purified lung ILC2s from Nb-infected control and basophil-depleted mice and cultured them in vitro for 24 h. This allowed us to remove the cells from the inhibitory presence of the NMB ligand. In the absence of NMB ligand, ILC2s from control and basophil-depleted mice produced similar amounts of IL-5 and IL-13 (Fig. 8a). However, addition of rNMB significantly reduced production of IL-5 and IL-13 by ILC2s from control mice, but had no impact on cytokine production by ILC2s obtained from basophil-depleted animals (Fig. 8a). Importantly, WT ILC2s treated with rNMB showed increased survival, suggesting that reduced cytokine levels were not a result of increased cell death (Fig. 8b). Similar results were also seen when WT ILC2s were sort-purified and stimulated with IL-33 and NMB simultaneously (Extended Data Fig. 10a-d). These data illustrate that NMB directly alters the activation state of ILC2s and suggest that basophils regulate this process.
The above data suggest that basophils prime ILC2s for NMB-mediated inhibition via their ability to regulate NMBR expression. However, whether basophils regulate NMBR expression in a direct or indirect manner remains unknown. To address this, we co-cultured ILC2s either alone or in the presence of activated basophils and monitored surface expression of NMBR as well as cytokine secretion and cell proliferation. As previously reported 14,15 , co-culture of ILC2s with activated basophils resulted in elevated secretion of cytokines but failed to promote changes in ILC2 proliferation (Extended Data Fig.  10e-g). Critically, ILC2s co-cultured with basophils also exhibited significantly increased surface expression of NMBR (Fig. 8c, d). Importantly, the ability of basophils to promote receptor expression was not a conserved feature of ILC2 activation, as IL-33 treatment had no effect on NMBR levels (Fig. 8e). Basophils are known to produce several important effector molecules including cytokines and lipid mediators that are capable of influencing the activation state of distinct immune cells including ILC2s 12,14,38,39 . Therefore, we also tested if the basophil-associated effector molecules IL-4 and prostaglandin E2 (PGE 2 ) are sufficient to increase NMBR expression. While IL-4 was found to have no effect, PGE 2 stimulation resulted in significantly increased surface expression of NMBR by sort-purified ILC2s (Fig. 8e). Collectively, these data suggest that basophils might prime ILC2s for NMB-mediated inhibition, in part, through their expression of PGE 2 .

Prostaglandin E2 and NMB cooperate to inhibit ILC2 activation
While, no changes in ILC2 viability were observed following NMB stimulation (Fig. 8b), it is possible that NMB inhibits ILC2s via its effect on cell proliferation and/or cytokine production. To evaluate whether rNMB affects proliferation, we stained sort-purified ILC2s with CellTrace™ Violet (CTV) and monitored cell division by employing a previously described culture system 40 . As expected, ILC2s showed strong proliferative capacity with approximately 40% of the cells undergoing 2-3 divisions and 15% of the cells undergoing 4-5 divisions over the 4-day culture period. As a positive control for inhibition, cultures were treated with PGE 2 41 . Consistent with previous reports, PGE 2 stimulation resulted in significantly decreased percentages of cells achieving between 2-5 divisions (Fig. 8f,g). Importantly, rNMB treatment alone showed no effect on the proliferation of ILC2s (Fig.  8f,g). Given the ability of PGE 2 to restrict ILC2 proliferation and our data demonstrating that PGE 2 appears to prime ILC2s for NMB-mediated inhibition (Fig. 8e), we sought to test if the two signals cooperate to simultaneously restrict both ILC2 proliferation and cytokine production. Similar to our previous data, ILC2s cultured with PGE 2 showed significantly increased expression of NMBR in our proliferation assays (Fig. 8h). Further, when PGE 2 and NMB were added together significantly less IL-5 and IL-13 were detected in culture supernatants, suggesting that the two signals cooperate to inhibit ILC2s via combined effects on proliferation and cytokine production (Fig. 8i).
While our data demonstrate that NMB-NMBR signaling inhibits ILC2 cytokine production, the molecular pathways that promote these changes remain to be defined. Therefore, we stimulated sort-purified ILC2s with either PBS or rNMB and performed RNA-sequencing analysis. Following rNMB stimulation, ILC2s exhibited significantly downregulated expression of the P2x purinoceptor 7 (P2rx7) a gene previously linked to ILC2 activation 42 (Fig. 8j, Extended Data Fig. 10h, i) (Supplementary table 2). To evaluate if P2rx7 inhibition phenocopies the effects of NMB treatment we treated sort-purified ILC2s with the P2rx7 inhibitor brilliant blue G 42 and monitored IL-5 and IL-13 levels. Consistent with the effects of rNMB treatment, inhibition of P2rx7 resulted in significantly reduced levels of IL-5 and IL-13 (Fig. 8k). Collectively, these data suggest that NMB may restrict ILC2 cytokine production via its inhibition of P2rx7.

Discussion
The findings presented above identify a previously unappreciated aspect of cross-talk between basophils and ILC2s. Moreover, these data suggest that communication between innate immune cells is required to promote neuro-immune interactions, necessary to maintain tissue integrity. While it is well-appreciated that robust type 2 cytokine production is vital to promote host protective responses to helminths 5,6 , our understanding of how this inflammation is restricted to prevent persistent tissue remodeling remains understudied. The data presented here identify a complex cellular and molecular network that is required to properly inhibit helminth-induced inflammation and identify a role for basophils in regulating these events.
While basophils have long been associated with type 2 cytokine-mediated inflammation, their diverse cellular functions have remained largely underappreciated. Although the dramatic basophilia that occurs following a Nb infection has been recognized for over 40 years 43,44 , the functions of Nb-elicited basophils remain controversial. Although several studies have reported that basophils are not required to promote Nb expulsion 20,21 , our data indicate that basophils enter the lungs after the parasitic larvae exit the tissue, suggesting that basophils are likely to participate in promoting tissue homoeostasis. Interestingly, recent studies have also determined that basophils infiltrate the lung during tissue development, where they serve important functions by programming alveolar macrophages 15,16 . Our data suggest that basophils might perform regenerative functions following an inflammatory response, in order to promote tissue homeostasis. A similar role for eosinophils in the lung has recently been established 45 , suggesting that type 2-associated granulocytes may perform tissue-protective functions in some contexts.
It is well established that communication between several innate immune populations is required to initiate potent type 2 cytokine responses. For example, basophils have been reported to activate ILC2s via their secretion of IL-4 12,14 . In response to these signals, ILC2s secrete vast amounts of IL-5 and IL-13 that elicit several events, such as eosinophil recruitment and mucus production by goblet cells. Nonetheless, whether innate immune cells also communicate to efficiently limit type 2 inflammation and prevent chronic tissue remodeling is not well understood. The studies presented here highlight a novel interaction between basophils and ILC2s that allows them to receive inhibitory signals induced by the neuropeptide NMB. It is possible that in the process of activating ILC2s via IL-4 secretion, basophils also prime ILC2s to receive regulatory signals required to limit the duration of their activation. This concept may be similar to the effects observed during T cell activation, in which TCR stimulation results in the induction of the inhibitory molecules PD-1 and CTLA-4 that become important negative regulators after the initial activation phase is completed 46,47 . Importantly, basophils, but not IL-4, were sufficient to induce NMBR on ILC2s, further supporting the notion that additional signaling pathways are initiated when basophils interact with ILC2s. PGE 2 is a well-defined lipid mediator known to be released by activated basophils 38 . Importantly, our data demonstrate that PGE 2 is sufficient to induce the expression of NMBR on ILC2s, although to a lesser extent than basophils. Interestingly, PGE 2 can inhibit ILC2 proliferation 41 and our data show that NMB selectively targets ILC2 cytokine production. Therefore, our data suggest a model where NMB and PGE 2 might cooperate to suppress ILC2 activation via their combined effects of proliferation and cytokine production. A similar concept is proposed by Nagashima et al. 27 where the neuropeptide CGRP shaped ILC2 functions by limiting NMU-induced proliferation and IL-13 secretion while supporting IL-5 production, suggesting that ILC2s may be regulated in specific manners that tailor to optimal outcomes. Although further studies are required to fully define the molecular actions of NMB, our data suggest that it might operate via its ability to regulate P2rx7 expression.
A growing body of evidence has demonstrated that highly coordinated interactions between the nervous and immune system are required to initiate host-protective responses against helminths 48 . However, whether similar communication events exist to limit helminthinduced inflammation remain to be fully defined. This is perhaps most evident in the context of ILC2 biology, where numerous mediators of activation, including NMU 23,24 , VIP 49 , and serotonin 50 , have been identified compared to a relatively limited number of inhibitors. Importantly, our studies identify a novel regulatory pathway in which NMB suppresses cytokine secretion by ILC2s, with basophils operating as the transition switch required for ILC2s to respond to NMB-mediated inhibition. Critically, our studies have also identified the expression of NMBR in several immune cells, including CD4 + T cells and alveolar macrophages, highlighting the need to further evaluate this critical pathway in other model systems. In summary, these data provide insight into the highly coordinated cellular and molecular events needed to allow for the necessary initiation, but carefully regulated persistence, of type 2 inflammation in the lung. NMBR-floxed mice were generated by Cyagen Biosciences Inc. Briefly, the gRNA to mouse Nmbr gene, the donor vector containing loxP sites flanking exon 2 of the Nmbr gene, and Cas9 mRNA were co-injected into fertilized mouse eggs to generate targeted conditional knockout mice. F0 founder animals were identified by PCR followed by sequence analysis. Positive offspring were then bred to wild type mice to test germline transmission and F1 animal generation. F1 mice were bred with wild type mice to generate more positive F2 animals, which were identified by PCR. NMBR floxed hemizygous mice were bred to generate homozygous offspring, which were then crossed with Vav-iCre hemizygous mice to target NMBR expression specifically on all hematopoietic cells. NMBR floxed homozygous Vav-iCre negative littermates were used as control.

Materials and Methods
All mice were maintained in specific pathogen-free facilities at the Rutgers New Jersey Medical School. All protocols were approved by the Rutgers Institutional Animal Care and Use Committee (IACUC), Protocol number 00968.

N. brasiliensis infection and pharmacologic treatments
Methods for maintenance, recovery, infection and isolation of Nippostrongylus brasiliensis (Nb) larvae were performed as previously described 53

Basophil transfer
WT mice were injected intravenously (i.v.) with a combination of 1µg mouse recombinant IL-3 (R&D systems) and 0.5μg of 〈-IL-3 antibody (BioLegend: clone MP2-8F8) in 200 μL of PBS 3 days prior to euthanasia as previously described 54 . At necropsy, single cell suspensions of spleens were prepared and basophil populations were sort-purified as described below using a FACSAriaII (BD Bioscience). Purity for all cell populations were determined to be 98% or greater. Then, 15,000 basophils were resuspended in 50μL of PBS and transferred into each mouse via intratracheal instillation at days 3, 4, 5, and 6 after Nb infection.

Intravascular in vivo flow cytometry stain
Intravascular in vivo staining protocols were performed similar to previously described 55 .
Briefly, mice were injected intravenously with 3μg of the basophil-specific fluorescentlabeled antibody anti-CD200R1 (clone OX110, eBioscience) diluted in 300μL of PBS, 5 minutes prior to euthanasia. Then, basophil populations in the lung tissue were analyzed by flow cytometry.

ILC2 in vitro cultures
Lung cells were isolated from WT or basophil-depleted mice infected with Nb 7 days prior. Then, ILC2 populations (CD45 + Lin -CD90 + CD127 + ) were sorted and 10,000 cells were cultured in the presence of 10 ng/mL of mouse recombinant IL-2, IL-7 (R&D systems), and vehicle (PBS) or 1 μg/mL of Neuromedin B for 20-24 hours. For some experiments, 10ng/mL of mouse recombinant IL-33, IL-4 (R&D systems), 100nM of Prostaglandin E 2 (PGE 2 ) (Cayman chemicals), or 1μM of brilliant blue G (Sigma-Aldrich) were added to ILC2 cultures. Cell viability was evaluated by 7-AAD (Biolegend Cat #420403) and Annexin V (Biolegend Cat #640920) double negative staining. Supernatant levels of IL-5 and IL-13 were then evaluated by ELISA. For intracellular staining, BD Golgi Plug™ Protein Transport Inhibitor (Cat. #555029) was added for the last 4 hours of incubation and cells were stained and analyzed as described above. NMBR expression was evaluated by flow cytometry analysis as indicated above.
For ILC2 basophil co-cultures, lung-resident ILC2 populations were sort-purified from WT or Rag2 -/mice after 7 days of Nb infection and cultured with basophils sort-purified from WT mice injected with 1μg of mouse recombinant IL-3 3 days prior. ILC2s and basophils were co-cultured for 24 hours in the presence of mouse recombinant IL-2, IL-7, and IL-3 (10ng/mL).

RNA isolation and quantitative real-time PCR analysis
RNA from sections of lung tissue was isolated by homogenization in TRIzol (Invitrogen) followed by phenol-chloroform extraction and isopropanol precipitation. cDNA was generated per standard protocol with Superscript reverse transcriptase (Invitrogen) and used as input for real-time PCR. Real-time PCR data were analyzed using the ΔΔCT method using SYBR Green chemistry (Applied Biosystems), with βNA fro serving as the endogenous housekeeping gene. All reactions were run on an ABI 7500 Fast Real-Time PCR System (Applied Biosystems). Samples were normalized to naïve controls. The following QuantiTech primer assays from Qiagen were used: Areg (

Global and single cell RNA-sequencing
Lung-resident ILC2s from control and basophil-depleted mice were sort-purified on live CD45 + CD3 -CD19 -CD11b -CD11c -NK1.1 -B220 -CD5 -Ter-119 -γδTCR -Fc γδRIα -CD90 + CD127 + cells from three biological replicates and pooled. Purity for all cell populations were determined to be 98% or greater. For bulk RNA-sequencing, RNA was extracted using Qiagen RNeasy plus™ micro kit (Cat. #74034), and amplified using Ambion MessageAmp II aRNA™ Amplification Kit (Cat. #AM1751). Illumina compatible libraries were generated using the NEBNext Ultra II kit (NEB E7645S) and sequenced using an Illumina NextSeq 550 in a single end 75bp read configuration. Bulk RNAsequencing analysis was performed in accordance with the NF-Core RNA-sequencing guidelines (version 1.4.2) 57 . The output reads were aligned to the GRCm38 genome using STAR, followed by gene count generation using featureCounts and StringTie [58][59][60] . Read counts were normalized and compared for differential gene expression using DESeq2 with significance at False Discovery Rate (FDR) adjusted p-value < 0.05 61 . For analysis of functional pathways, the Database for Annotation, Visualization, and Integrated Discovery (DAVID) platform 62 was used.
For single-cell RNA-sequencing, 10,000 sort-purified ILC2s were processed using the 10X Genomics Chromium™ Controller. Cell suspensions were loaded onto the Chromium Single Cell A Chip for cell lysis and barcoding. RNA from individual cells was reverse transcribed and sequencing libraries prepared using the Chromium™ Single Cell 3' Library Kit v2, following the manufacturers protocol. Samples were sequenced using an Illumina NextSeq 550 with standard 10X Genomics Configuration (26bpx98bp). After sequencing, raw bcl files were processed using the cellranger mkfastq command for sample demultiplexing and conversion to .fastq files, followed by cellranger count for cell barcode and UMI deconvolution as well as mapping to the respective reference genome. Processed digital gene expression matrices were imported into R studio for analysis using the Seurat package. Samples were aligned along common sources of variation and compared using canonical correlation analysis to identify unique clusters of cells within the samples. Marker genes for each sample and cluster were identified and used for generation of downstream plots within the Seurat package. All packages are maintained to be best in class and are regularly updated to their most recent release.

Pulse oximetry
Oxygen saturation was evaluated with the MouseOx Plus ® (Starr Lifesciences Corp) following manufacturer's instructions. Briefly, the hair around the thigh was removed 1 day before Nb infection. Then, mice were anesthetized with 5% isoflurane and oxygen saturation was monitored using the thigh sensor for an interval of approximately 5 minutes. This time point was used to collect representative and error-free data.

Histology
Lung sections were fixed in formalin 10% buffered for at least 3 days, followed by processing in a Leica ASP300 tissue processor following manufacturer's instructions. Tissue blocks were paraffin-embedded using a Leica EG1150H Embedding Module and then sliced into 5μm sections using a Leica RM2235 rotatory microtome. Tissue sections were then deparaffinized, hydrated and stained with hematoxylin & eosin (H&E) or periodic acid-Schiff (PAS) methods. Images were acquired with a Keyence BZ-X710 All-in-One fluorescence microscope (Keyence, Osaka, Japan) and analyzed using the Keyence BZ-X Viewer version 01.03 software.

Statistics
Results are shown as mean ± standard error of the mean. P<0.05 was considered as significantly different. Statistical analysis was performed using Student's t-tests in GraphPad Prism version 8.

Reporting Summary.
Further information on research design is available in the Nature Research Life Sciences Reporting Summary linked to this article.

Extended Data
Extended Data Figure 1. Basophils limit helminth-induced pulmonary inflammation.