The animal protocol for this study was approved by the National Jewish Health IACUC. We used B6.Cg-Nfkb1tm1Bal/J from the Jackson Laboratory (JAX stock #006097). This strain is commonly known as p50- or Nfκb1 knockout (Nfκb1-/-). We used littermate, and in select experiments, C57BL/6 as wild-type controls.

We used the Alternaria alternata (Alt) allergen extract. Mice were sensitized to Alt as shown in Figure 1 . Briefly, Alt (10 μg/dose), was administered in 20 ul volume intranasally on alternate days 3x a week for 3 consecutive weeks unless otherwise stated. The mice were then rested for 3 weeks. In week 7 they were given a recall challenge with a subthreshold dose of the sensitizing allergen on 3 consecutive days. The subthreshold recall dose was 2.5 ug/mouse. The mice were sacrificed 3 days later.

The measurement of airway hyperreactivity in response to methacholine by Flexivent was described in details previously (13). Briefly, mice were anesthetized with ketamine (180 mg/kg), xylazine (9 mg/kg), and acepromazine (4 mg/kg). After loss of foot-pad pinch reflex, a tracheotomy was performed and the mouse was attached via an 18-gauge cannula to a small-animal ventilator with a computer-controlled piston (Flexivent; Scireq) (flexiVent Fx; SCIREQ, Montreal, Quebec, Canada). After performing initial calibrations (cylinder pressure channel and nebulizer calibration), we conducted dynamic tube calibration and used a default program called QuickPrime 3 (version 7) for measurement of airway resistance in response to methacholine. This program uses prime perturbations, which are a family of complex forced oscillation perturbations at a frequency greater than and less than the subject’s ventilation frequency (1-20.5 Hz). The amplitude of the oscillatory signal is preset to a volume that is slightly smaller than the subject’s tidal volume (0.2 mL). Volume and pressure signal are recorded during a measurement, and the flow signal is derived from the volume. The foregoing allows calculation of Newtonian resistance, tissue damping, tissue elastance, and hysteresivity. Resistance measurements were taken to establish the baseline for total lung resistance and at each methacholine dose. Group averages were expressed as the fold increase over baseline resistance (mean ± SEM) (13).

Paraffin embedded lungs were sectioned and stained with hematoxylin and eosin (H&E) for morphometric analysis, PAS staining. For mucus and Mason’s trichrome for collagen deposition. We measured the entire peribronchial inflammatory area (infiltrate area). We also measured the perimeter of the basement membrane (BM) of the corresponding bronchus. We presented the data as the infiltrate area/μM BM. Images were acquired on a Nikon Eclipse TE2000-U microscope using 20x dry lenses at room temperature through a Diagnostics Instruments camera model #4.2 using the Spot software 5.0. H&E, PAS and trichrome sections were mounted using Permount medium. Images were adjusted for brightness and contrast to improve viewing (13)

Paraffin-embedded lung sections (4-µm thickness) were used for this study. Images were taken under polarized light using an upright dry 40× objective. Tissues were deparaffinized and after antigen retrieval, permeabilized with 0.4% triton X-100. Tissues were blocked with 10% BSA and maintained in PBS + 5% BSA + 0.4% triton X-100 throughout antibody treatments. Primary antibodies were incubated at 4°C overnight and secondary antibodies were incubated for 1 hr at room temperature. Primary antibodies used (1:200 dilution) include rabbit anti-NFκB p65 (#8242, Cells Signaling Technology); mouse anti-cRel (#MA5-15859, Thermo Fisher Scientific); rabbit anti-NFκB1 (#13586, Cells Signaling Technology); mouse anti-NFκB1 (#NBP2-66976, Novus); rabbit anti-RUNX1(#ab229482, Abcam); goat anti-IL33 (#AF3626 R&D); mouse anti-ICAM1 (#sc-1511, Santa Cruz Biotechnology, Inc); and mouse anti-CD3 ((# sc-7296, Santa Cruz Biotechnology, Inc). Goat anti-rabbit and anti-mouse IgG-A594 or IgG-A488 were used as secondary antibodies (1:200 dilution at RT). For anti-goat, rabbit anti-goat IgGA-647 was used as secondary antibody (1:200 dilution at RT). DAPI was used for nuclear counterstaining. The ProLong Gold antifade reagent was used as mounting media. Mount slides were examined with a Leica DM6000 B. Slide Book 6 (3i) was used for analysis and capturing images.

Mouse lungs were perfused with saline and then subjected to mechanical mincing followed by digestion at 37°C for 45 minutes in RPMI with 10% FBS, 1% penicillin/streptomycin and collagenase Type I (1mg/mL) (Worthington # LS004197) as described previously (13). Isolated cell suspensions were agitated at room temperature for 10 minutes in RPMI with 100U/mL DNAse I prior to filtration through 40μm filters and red blood cell lysis. Single cell suspensions were either subsequently cultured in RPMI with 10% FBS, 1% penicillin/streptomycin at 37⁰C in CO2 incubator overnight or used immediately for staining for flow cytometry and analysis, depending on the experiment and schedules (13).

The mouse lung single cell suspension was blocked with an anti-FcR blocking reagent for 10 min (minutes) at 4⁰C (Miltenyi Biotec; # 130-092-575) before staining with a fixable viability dye. Cells were then stained with surface receptor antibodies at 4⁰C for 30 min (cells were washed twice with staining buffer (PBS plus 1% BSA between each staining). Then we fixed the cells with 4% paraformaldehyde for 15 min at 4⁰C, washed them with the staining buffer (PBS plus 1% BSA), and incubated with the permeabilization buffer (PBS [pH 7.4] plus 1% BSA plus 0.1% saponin) at 4⁰C for 20 min. After centrifugation, we resuspended the cells in the permeabilization buffer and incubated with antibodies against the intracellular proteins (cytokines and transcription factors) at 4⁰C for 30 minutes. We washed the cells twice with the permeabilization buffer, resuspended in the staining buffer, and maintained at 4⁰C until flow analysis. For intercellular cytokines, we added monensin (2 mmol/L) to the cells and cultured for 4 hours before staining

Most of the fluorophore-conjugated antibodies used for flow cytometry were purchased from Biolegend, Inc. (San Diego, CA). Others were from eBioscience or R&D, Inc., unless otherwise stated. ILC cells were stain with FITC or BV605-labelled anti-CD45.2 (clone 104), pacific blue or Alexa Flour 700 -labeled lineage marker antibodies (CD3, Ly-6G/Ly-6C, CD11b, CD45R/B220, TER-119/Erythroid cells, pacific blue or Alexa Flour 700 FcϵR1α (Biolegend # 134314 or 134324), PerCP-Cy5.5-conjugated anti-CD25 (eBioscience; clone PC61.5), pacific blue or Alexa Flour anti-mouse NK-1.1 Antibody (Biolegend # 108722 or 156512)., APC or PE labelled anti-IL5 (TRFK5), and PE-Cy7 or PE labeled anti-IL13 (eBioscience; clone eBio13A). For eosinophil and neutrophil: Clone #245707), PerCP/Cy5.5 anti-mouse Ly-6G (Biolegend # 127616; clone1A8), PE/Cy7 anti-mouse CD11c (Biolegend # 117318; clone N418), Brilliant Violet 421™ anti-mouse/human CD11b (Biolegd# 101236; cloneM1/70), PE anti-mouse CD193(Biolegend # 144506; clone J073E5), Alexa Fluor^® 647 Rat Anti-Mouse Siglec-F (BD Pharmingen™# 562680; clone E50-2440). PerCP-eFluor^® 710 anti-GATA3 Antibody (eBioscience # 46-9966-42) and Brilliant Violet 605™ anti-mouse NK-1.1 Antibody, Fixable Viability Dye eFluor™ 780 (#65-0865-14 from eBioscience) and Zombie Aqua™ Fixable Viability Kit (#423102 from Biolegend) used for detection of cell viability. Stained cells were analyzed using the LSR Fortessa cell analyzer (BD). Flow data were analyzed with FlowJo version 10.0.7 software (Tree Star).

Total RNA was isolated from frozen lung samples using Trizol (Invitrogen). cDNA was synthesized using the Verso cDNA synthesis kit (Thermo Scientific # AB-1453/B) according to manufacturer’s instructions as described previously (13). Gene specific PCR products were amplified using the qPCR SYBR Green Rox mix (ThermoScientific # AB-4162/B) and primers outlined in online repository Table S1 . Primers were designed using the Applied Biosystems 7000 Sequence Detection System software. The levels of target gene expression were normalized to 18S expression using the 2^-ΔCt method. The primer list is given in Table S1

Lysates for extraction of protein and for immunoprecipitation (IP) were prepared using RIPA Lysis and extraction buffer # 89900 and Pierce IP Lysis buffer # 87787; Thermo Fisher Scientific, respectively, according to the manufacturer protocol. Protein concentration was determined by the BCA method (Pierce™ BCA Protein Assay Kit # 23225; Thermo Fisher Scientific). An aliquot of 20 µg the lysates was used for SDS– polyacrylamide gel electrophoresis (SDS-PAGE) and analyzed by immunoblotting. Blot was incubated overnight at 4°C with respective antibody; rabbit anti-NFκB p65 (#8242, Cells Signaling Technology); mouse anti-cRel (#MA5-15859, Thermo Fisher Scientific); rabbit anti-NFκB1 (#13586, Cells Signaling Technology); mouse anti-NFκB2 (# NBP2-66977, Novus); rabbit anti-RUNX1(#ab229482, Abacm); and detected using an HRP conjugated IgG antibody. For IP 100 µg of the lysates was incubated overnight at 4°C with an immunoprecipitating antibody (RUNX1 # LS-B13948; LSBio, NFκB1 (# 13586; Cell Signaling Technology) and β-actin (#12262; Cell Signaling Technology) along with an appropriate isotype control antibody. Protein A/G PLUS-Agarose (sc-2003; Santa Cruz Biotechnology, Inc) was added and kept at 4°C on a rotating platform for 2 h. Thereafter, the immune complexes were isolated, and separated by SDS—PAGE.

IL5 (R&D#DY405-05) and IL13 (R&D#DY413-05) in the bronchoalveolar lavage fluid were measured by ELISA kits as per manufacturer’s instruction.

Statistical analyses were performed, and data figures were prepared with GraphPad Prism software (version 6; GraphPad Software, San Diego, Calif). Statistical significance was analyzed by t test or ANOVA.

To understand the role of NFκB1 in allergen-induced memory ILC mediated asthma, we treated C57B/6 mice with Alternaria (Alt) and saline (Sal) as described previously (1). According to this protocol ( Figure 1A ), mice were intranasally exposed to 10 ug/dose of Alt or Sal 3 days a week for 3 consecutive weeks, rested for 3 weeks and then given a recall challenge with a subthreshold 2.5 ug/dose of Alt on 3 consecutive days. AHR and inflammatory indices were measured 3 days later. We found that Alt/Alt treated mice had increased AHR, and increased frequency of bronchoalveolar lavage (BAL) lymphocytes, eosinophils and neutrophils, and lung eosinophils as compared to Sal/Alt mice ( Figure 1B ). Cytokine measurement in BAL showed elevated levels of IL5 and IL13 ( Figure 1C ). Lung histology showed increased levels of peribronchial and perivascular inflammation, mucus-producing goblet cells, and airway remodeling in Alt/Alt mice ( Figures 1D, E ). Flow cytometric analysis of the lung cells showed an increased number as well as frequency of total ILCs (CD45+Lin-CD25+NK1.1- FcϵR1α-), ILC2s (CD45+Lin-CD25+NK1.1- FcϵR1α-ICOS+ ST2+) as well as type 2 cytokine (IL5 and IL13)+ ILC2s ( Figure 1F ; Figure S1A ). The gating strategy for the flow analysis is shown in Figures S1B–F . NFκB1 was previously shown to be involved in regulation of ILC2, CD4+ ILC3, and NCR+ ILC3 (14). We checked its expression by flow cytometry in Alt/Alt and Sal/Sal treated mice. p105/50 was highly expressed in ILC2s and minimally in non-ILCs (Lin+ CD45+ cells) ( Figures 1G–I ). We checked p105/50 expression in NK, ILC1, ILC3, epithelial and CD4 cells. The frequency of p105/50+ cells was the highest in ILC2s as compared to other cells ( Figures S2A–D ). Next, we examined nuclear localization of p105/50 in the lung tissue by immunofluorescence staining. Alt/Alt mice showed a higher level of nuclear localization of p105/50 than Sal/Alt mice ( Figure 1J ). We confirmed p105/50 expression in lung cells from Alt/Alt mice by western blot ( Figure 1K ). This data indicated that increased AHR and type-2 inflammation in allergen treated mice were associated with increased expression of NFκB1.

The role of NFκB1 in memory ILC2-mediated asthma is unknown. To address this question, we designed the following experiment. We sensitized Nfκb1+/+ (littermate) and Nfκb1-/- female mice intranasally with Alt according to Figure 1A . Nfκb1-/- mice sensitized to and recalled with Alt (Alt/Alt) had negligible AHR and reduced BAL lymphocytes, eosinophils and neutrophils as compared to Nfκb1+/+ mice ( Figure 2A ). Alt/Alt treated Nfκb1-/- mice had increased CD11b+Ly6G+ neutrophils and decreased Siglec F+ CCR3+ eosinophils in the lung as compared to Nfκb1+/+ mice (as measured by flow cytometry) ( Figure 2B ; Figure S3A ). Nfκb1-/- mice had lower levels of IL5 and IL13 in BAL (ELISA) ( Figure 2C ). Lung histology showed reduced levels of peribronchial and perivascular inflammation, and mucus-producing goblet cells ( Figures 2D, E ). Airway remodeling was similar in Nfκb1-/- and Nfκb1+/+ mice ( Figures 2D, E ). We examined the expression of mRNA for select type-2 inflammation associated genes. The expression of mRNA for IL5 and IL13 was decreased in Nfκb1-/- mice ( Figure 2F ). The mRNA for eosinophil-associated ribonuclease A family member 2 (Ear2) and the mucin gene Muc5ac were lower while that for Muc5b and the neutrophil elastase were unchanged in knockout mice ( Figures 2G, H ). Flow cytometric analysis of the lung cells showed reduced frequency of total ILCs (CD45+Lin-CD25+NK1.1- FcϵR1α-), ILC2s (CD45+Lin-CD25+NK1.1- FcϵR1α-ICOS+ ST2+) and type 2 cytokine (IL5 and IL13)+ ILC2s ( Figure 2I ). The gating strategy for the flow analysis is shown in Figure S3B . We repeated this experiment using a group of male mice. We observed similar trends with Nfκb1-/- mice ( Figures S3C–G ). The results suggested that Nfκb1 was important for the effector phase of memory ILC2-driven asthma.

Next, we examined the effect of Nfκb1 deletion on Tregs, ILC3 and CD4 T cells. We previously reported that our chronic asthma model was associated with heightened numbers of CD4 T cells and CD4+ nTregs, and that CD4 T cells contributed to the increased magnitude of airway inflammation (13, 15). CD4+Foxp3+CD25+ Tregs, CD127+ST2+ILC2s and RoRyt+CD127+ILC3 but not CD4 T cells were reduced in Nfκb1-/- ( Figure 3A ). Likewise, IL5 and IL13+ ILC2s but not CD4 T cells were reduced in Nfκb1-/- mice ( Figure 3B ). Note that the frequency of IL5+ and IL13+ CD4 T cells was low in control wild type mice in our asthma model. We checked the total number of CD4+ T cells in the lung and found ~ 50% less CD4+ T cells in Nfκb1-/- mice ( Figure 3C ). Next, we asked whether the development of CD4 T cells and ILC2s was impaired in Nfκb1-/- mice. To address this question we studied naïve Nfκb1+/+ and Nfκb1-/- mice. The frequency of CD4+ T cells and CD25+, ST2+, ICOS+ and GATA3+ ILC2s was similar in both mouse strains. ( Figure 3D ; Figure S4A ).

GATA3 is a signature transcription factor for type-2 T cells and ILCs. GATA3 expression and its mean florescence intensity (MFI) were similar in ILC2s and CD4 T cells from Nfκb1+/+ and Nfκb1-/- mice ( Figure 3D ; Figure S4B ), which did not explain reduced cytokine production by ILC2s in Nfκb1-/- mice. Next, we studied RUNX1, another transcription factor that contributes to ILC2 cytokine production (16). Nfκb1-/- mice showed reduced expression of RUNX1 ( Figure S4C ).

TSLP, IL25 and IL33 are three major upstream cytokines for the type-2 immune response, and they are frequently elevated after an allergen exposure. We did not observe any significant difference in mRNA for IL25 and TSLP ( Figure S4D ). TSLPR+ ILC2s were reduced in Nfκb1-/- mice ( Figure S4E ). IL25R+ (IL17RE+) ILC2s were not detected (data not shown) by flow in our model. In contrast to IL25 and TSLP, IL33 was reduced at the mRNA and the protein level in Nfκb1-/- mice ( Figures S4F, G ). ICAM1 regulates inflammatory cell influx into the tissue. Since Nfκb1-/- mice had reduced inflammation, we studied ICAM1 expression. ICAM1 expression was reduced in Nfκb1-/- mice ( Figure S4H ). The foregoing findings suggested that Nfκb1 was important for induction of select transcription factors and adhesion molecules.

Next, we examined the importance of NFκB1 in ILC2 memory formation. We intranasally sensitized Nfκb1+/+ (littermate) and Nfκb1-/- female mice with Alt and studied them 3 weeks later without a recall allergen challenge and thereby, avoiding the elicitation of the effector function as shown in Figure 4A . Both strains (Nfκb1+/+ and Nfκb1-/-) had the same level of AHR and Siglec F+ CCR3+ eosinophils in the lung ( Figure 4B ; Figure S5A ). Note that the amplitude of AHR and eosinophilic inflammation was much lower in the absence of the recall allergen challenge. Both strains showed a similar frequency of total ILCs as well as ILC2s cells. However, Nfκb1-/- mice had lower frequency of IL5+ and IL13+ ILC2s ( Figures 4C, D ; Figure S5B ). The decreased expression of IL5 and IL13 in BAL was confirmed by ELISA ( Figure S5C ). We checked the frequency of CD4 T cells and type-2 cytokine+ CD4 T cells, which were, surprisingly, similar in both strains ( Figure 4E ; Figures S5D, E ).

Next, we examined genes involved in ILC2 memory-associated programs (1)—the gene repression program (Zeb1, Nr4a2, Bach2, JunD, Fra1 and Fra2) ( Figures 4F–H ) and the preparedness program (Fhl2, Mpp7, Stat6, and Srebf2) ( Figures 4I, J ). We studied them at two different time points—after sensitization but before recall in week 6, and after recall in week 7. Most of these genes were elevated before recall during memory formation ( Figures 4F–J ). Nfκb1-/- mice had heightened expression of these genes regardless of the timing of measurement—before or after recall. The results suggested that Nfκb1 was a negative regulator of ILC2 memory-associated gene repression and preparedness programs.

We performed immunofluorescence staining for p65, c-Rel and RelB on the lung tissue from Nfκb1-/- and Nfκb1+/+ mice. Both strains showed a similar frequency of p65+ cells but Nfκb1-/- mice had increased p65 nuclear localization ( Figures 5A–C ). RelB immunostaining was negative in both groups (not shown). c-Rel immunostaining was detected in both groups. However, Nfκb1-/- had less c-Rel+ cells and reduced nuclear localization when compared with Nfκb1+/+ ( Figures 5D–F ). Nfκb1-/- mice showed higher levels of nuclear localization of p65 but surprisingly, no inflammation. To investigate this further, we performed double immunofluorescence staining for CD3 and p65 or p105/50. p65+ cells were mostly CD3+ whereas p105/50+ cells were mostly negative for CD3 ( Figures 5G, H ). Similar to p65, c-Rel+ cells were mostly CD3+ ( Figures S6A, B ). We did western blot of the lung tissue from Alt/Alt mice for p105/50, p100/52, p65, c-Rel and RUNX1. The expression of RelB, c-Rel, and 105/50, was mostly absent but that of p65 was marginally altered in the lung tissue from Nfκb1-/- mice ( Figure 5I ). The expression of RUNX1 and p100/52 was reduced. The foregoing data suggested a differential expression pattern of NFκB family members by ILC2s and CD3 T cells. NFκB1 positively regulated the expression of most of the NFκB family members except that of p65.

RUNX1 expression by ILC2s was low in Nfκb1-/- mice ( Figure S4C ). We compared the expression of RUNX1 between Lin+ and Lin- cells from Nfκb1 sufficient mice. The frequency of RUNX1+ cells was much higher in the Lin- as compared to the Lin+ cell population ( Figures 6A, B ). We did not observe any difference in RUNX1 expression in Lin+ cells between the Nfκb1+/+ and Nfκb1-/- mouse strains ( Figures S6C, D ). We studied co-localization of p105/50 and RUNX1 by immunofluorescence staining in the lung tissue from Alt/Alt sensitized Nfκb1+/+ and Nfκb1-/- (as a control) mice. RUNX1 and p105/50 were co-localized in Nfκb1+/+ mice, which was expectedly absent in Nfκb1 -/- mice ( Figure 6C ). The heterodimer of RUNX1 and p105/50 functions as a transcriptional activator (17). To examine if heterodimers were present, we performed co-immunoprecipitation experiments with p105/50 and RUNX1. RUNX1 co-precipitated with p105/50 and conversely, p105/50 co-precipitated with RUNX1 ( Figures 6D, E ). We speculate that NFκB1 positively regulated the effector phase of memory ILC2-induced asthma by heterodimerizing with RUNX1.