Type 2 inflammation is found in most forms of asthma, which may co-exist with recurrent viral 23 infections, bacterial colonization, and host cell death. These processes drive the accumulation of 24 intracellular cyclic-di-nucleotides such as cyclic-di-GMP (CDG). Group 2 innate lymphoid cells (ILC2s) 25 are critical drivers of type 2 lung inflammation during fungal allergen exposure in mice; however, it is 26 unclear how CDG regulates lung ILC responses during lung inflammation. Here, we show that CDG 27 induced early airway type 1 interferon (IFN) production and dramatically suppressed both lung 28 proliferating CD127+ST2+ ILC2s and Alternaria-and IL-33-induced lung inflammation. Further, 29 transcriptomic analysis of CD127−ST2− Thy1.2+ ILCs, which were expanded and activated by CDG, 30 revealed an ILC1 signature. CDG administration led to accumulation of IFNγ+ and T-bet+ ILC1s, as well 31 as neutrophilia, independent of IL-18R, IL-12, and STAT6 but dependent on stimulator of interferon genes 32 (STING) and partially dependent on type 1 IFN signaling. Thus, CDG potently suppresses ILC2-driven 33 lung inflammation and promotes ILC1 responses. These results suggest potential therapeutic modulation 34 INTRODUCTION plasticity (26, 29), we further investigated the impact of CDG on ILC identity, activation, and 148 proliferation. CDG decreased lung ILC2 expression of the canonical ILC2 master type 2 cytokine 149 regulator GATA-3 ( Figure conversion of ST2+ ILC2s to ILC1s during lung inflammatory responses to influenza has been shown to 172 be promoted by 31) . Thus, we investigated whether IL-12 and IL-18 signaling were 173 required for the observed granulocytic shift after addition of CDG to Alt. Surprisingly, we found that the 174 levels of airway eosinophilia and neutrophilia, and frequencies of ILC2s and ILC1s, remained constant in 175 mice lacking IL-12 ( Figure 3H , I, L, Supplementary Figure 3D , E) and IL-18R ( Figure 3J , K, M, 176 Supplementary Figure 3F , G). Collectively, these results suggest that CDG does not regulate ILC2 to 177 ILC1 responses through IL-12 or IL-18. 178
Given the robust airway IFN accumulation induced by CDG, we next investigated whether STING 181 (the stimulator of interferon genes) was required for suppression of type 2 lung inflammation and ILC2 182 responses. Strikingly, through comparison of wild type and STING deficient (Tmem173−/−) mice, we 183 found that removal of STING completely rescued CDG-driven abrogation of Alt-induced lung 184 eosinophilia and fully abrogated CDG-induced lung neutrophilia ( Figure 4A , B). Moreover, lung ILC2 185 function was completely restored in the absence of STING ( Figure 4C ), and CDG-driven ILC1 IFNγ 186 induction was entirely STING-dependent ( Figure 4D ). 187
A recent study demonstrated that STAT6 was required for STING-dependent chemokine 190 production (32). Therefore, we sought to determine whether STAT6 was required for neutrophil 191 accumulation in lung tissue following CDG challenge. Consistent with previous reports, Alt-induced 192 eosinophilia was highly dependent upon STAT6 ( Figure 4E ) (25). Notably, we found that residual lung 193 eosinophil levels following CDG administration were further reduced in the absence of STAT6. Unlike 194 eosinophils, neutrophils accumulated in the airway independent of STAT6 ( Figure 4F ). These results demonstrate that while STAT6 regulates innate type 2 lung inflammation, likely through chemokine 196 expression, it has no role in the STING-induced effects on neutrophil accumulation (25, 33). 197 198 Type 1 IFN signaling is partially required for CDG induced immunomodulation 199
Type 1 IFNs are thought to be the major products of stimulator of interferon genes (STING) 200 activation. To confirm that CDG-induced type 1 IFN production was responsible for the observed 201 immunomodulation, we compared type 1 IFN signaling deficient (Ifnar1−/−), wild type, and Tmem173−/− 202 mice. Surprisingly, Ifnar1−/− mice did not phenocopy Tmem173−/− mice. Unlike STING deficiency, 203 removal of type 1 IFN signaling only partially rescued CDG-induced abrogation of BAL eosinophilia 204 ( Figure 5A ) and did not fully prevent CDG-induced BAL neutrophilia ( Figure 5B ). Moreover, as in wild 205 type mice, the frequency of ILC1s was significantly increased following CDG challenge in mice deficient 206 in type 1 IFN signaling; whereas, the frequency of ILC2s remained constant in Ifnar1−/− mice, as in those 207 lacking STING ( Figure 5C ). Thus, type 1 IFNs are only partially required for CDG-induced 208 immunomodulation, indicating the observed shifts in peribronchial inflammation and ILCs depend on an 209 alternative mediator downstream of STING. Asthma is largely a type 2 inflammatory airway disease and is associated with bacterial and viral 222 infections, mitochondrial stress, and host cell death, which all lead to the accumulation of the danger-223 associated cyclic-di-nucleotides (5-8, 34). ILC2s promote type 2 inflammation in experimental asthma 224 models and likely contribute to airway inflammation and hyperresponsiveness in humans (35). Here, we 225 investigated how in vivo administration of the cyclic-di-nucleotide CDG regulates ILC2s and innate type 226 2 inflammation. Our novel findings show that intranasal CDG challenge nearly abolished Alt-induced 227 airway eosinophilia but promoted neutrophil accumulation. Congruently, CDG drove contraction of IL-228 5+ and IL-13+ lung ILC2s and expansion of IFNγ+ ILC1s. Mechanistically, we found that CDG 229 modulated ILC responses downstream of Alt-induced IL-33 release and that the effect of CDG on airway 230 inflammation and ILC function was entirely STING-dependent and partially type 1 IFN-dependent. 231 STING's involvement in pathogen responses, as well as activation by cytosolic mitochondrial DNA (from 232 damaged mitochondria) and autophagy, could represent a critical, common pathway in the development 233 of mixed airway inflammatory responses often found in asthma (8, (36) (37) (38) (39) (40) . 234
While type 1 IFNs are the major products of STING activation, STING agonism has also been 235 shown to induce the production of several inflammatory mediators including type 3 IFN, IL-1b, 236 TNFa, CCL2, . In addition to elevated levels of type 1 IFNs, we observed an increase 237 in type 3 IFN following CDG administration, though it failed to reach significance. Type 1, 2 and 3 IFNs 238 are known to inhibit ILC2 function and restrict type lung 2 inflammation (23, 24, 45, 46) , however the 239 effect on development of ILC1 accumulation by IFNs is not well studied. Taken together with recent 240 reports, our findings suggest a model in which CDG drives STING-dependent early production of type 1 241 and 3 IFNs, which suppress ILC2s and promote ILC1 IFNγ that further suppresses ILC2s. Because the 242 effect of CDG on Alt-induced inflammation we observed was only partially dependent on type 1 IFN signaling, 243 non-IFN mediators downstream of STING likely contribute to CDG-induced immunomodulation. 244 previous report in which CDG was administered in an adaptive Th2 cell-driven chronic asthma model, 246 though ILC changes were not assessed (47). Further, the ILC compartmental changes we observed mirror 247 influenza virus-induced ILC2 to ILC1 plasticity described in a recent study (30). However, that study 248 demonstrated that IL-12 and IL-18 drove the attenuation of type 2 cytokine production, whereas we found 249 that CDG abrogated type 2 inflammation independent of IL-12 and IL-18 signaling and dependent on 250 STING and type 1 IFNs. Collective, these findings indicate that there are multiple ways to induce an 251 ILC2 to ILC1 shift in the lungs. 252
Interestingly, a recent investigation into the role of STING in the gastrointestinal mucosa revealed 253 a nearly two-fold reduction in ILC2 frequency and IL-4 and IL-13 levels, and a three-fold increase in ILC1 254 frequency, in STING deficient mice (48). Conversely, we found markedly increased airway ILC2s and 255 type 2 cytokines, and decreased ILC1s and type 1 cytokines, in STING deficient mice following Alt and 256 CDG challenge. Such highly divergent findings suggest the presence distinct, tissue-specific mechanisms 257 by which STING controls ILC responses. These results are consistent with recent work demonstrating 258 tissue specific ILC responses (49). 259
The findings we report here might be leveraged for developing treatments to induce early, local 260 control of respiratory viruses, including SARS-CoV-2, in which type 1 and 3 IFN control of virus may be 261 critical for prevention of late complications in severe disease (19, 21) . RNA viruses, such as 262 coronaviruses, induce STING downstream of RIG-I and MAVs signaling (50). Importantly, however, 263 coronaviruses have evolved mechanisms for inhibiting STING activation. For example, coronaviruses 264 encode proteases that antagonize STING and prevent downstream IFN production (51, 52). Interestingly, 265 bats, which harbor coronaviruses yet show no signs of immunopathology, constitutively express IFN and 266
were recently shown to possess a distinct STING from that of other mammals (53). When bat STING 267 protein was humanized with a single amino acid substitution, viral tolerance was lost, suggesting a critical 268 role for STING activation and IFN expression in controlling viral replication. Additionally, patients with severe COVID-19 have impaired type 1 IFN responses (21), and early administration of the type 1 270 interferon IFN-α2b led to reduced in-hospital mortality in . Collectively, our 271 work, coupled with these reports, suggests that CDG, or other human STING agonists, may be a potential 272 low-cost, local respiratory therapeutic for early treatment of COVID-19 following SARS-CoV2 infection 273 through broad generation of anti-viral type 1 and 3 IFNs. 274
Our study is limited in that mechanisms downstream of STING that account for airway neutrophil 275 and ILC1 accumulation remain unclear. Consistent with previous reports, we showed that lung 276 eosinophilia is dependent on STAT6 (25). A prior study showed that STAT6 was required for STING-277 mediated chemokine production and immune cell recruitment (32); however, in our study, CDG-induced 278 airway neutrophil accumulation occurred independent of STAT6, which suggests the existence of an 279 alternative pathway by which immune cells accumulate in the presence of STING agonism. Future studies 280 investigating this distinct mechanism are warranted. 281
In summary, our work demonstrates that CDG drives STING-dependent IFN production, ILC1 282 activation and accumulation, as well as ILC2 suppression and abrogation of innate type 2 innate airway 283 inflammation. The knowledge set forth in this report is critical for understanding how allergic airway 284 disease pathogenesis may be impacted by lung insults due to cellular stress, bacterial infection, or viral 285 infections such as SARS-CoV2. In vivo Alternaria models 306 WT, gene knockout, and cytokine reporter mice were challenged intranasally with PBS, 5ug CDG, 307 50ug of Alt extract (Greer, lot number 299382), or 5ug CDG and 50ug Alt extract in 40uL every 24 hours 308 for 3 days and euthanized 24 hours following the third challenge for BAL collection and lung tissue 309 harvest. A subset of experiments was performed with only the latter two experimental groups. Further, 310
in select experiments, Alt was replaced with exogenous IL-33 (500ng). For experiments investigating 311 early cytokine release, mice were euthanized 3 hours following the first challenge. BAL was performed 312 with 2% BSA in PBS; the first draw was 500uL and draws 2-5 were 600uL. BAL was centrifuged at 1500 313 rpm for 5 minutes at 4C and the supernatant was stored at −20C for ELISA and cells were counted and 314 phenotyped using flow cytometry. Lungs were digested using the Mouse Lung Dissociation Kit (Miltenyi 315 Biotec) according to the manufacturer's protocol, filtered with a 40um mesh, and cells were counted and 316 phenotyped using flow cytometry. 317 PBS and counted on a Novocyte (Acea Biosciences). Cells were incubated with an unconjugated mAb to 321 CD16/CD32 for 10 minutes at 4C to block non-specific Fc receptor binding and then incubated for 30 322 minutes with fluorochrome conjugated antibodies at 4C. All antibodies were purchased from BioLegend 323 unless otherwise noted. To identify eosinophils (CD45+Siglec-F+CD11c−) and neutrophils (CD45+GR- For ILC cytokine staining, lung cells from WT and knockout mice were incubated for 3 hours at 337 37C with a phorbol 12-myristate 13-acetate, ionomycin, brefeldin A, and monensin cell stimulation 338 cocktail (ThermoFisher) in RPMI 1640 media supplemented with penicillin/streptomycin, 10% FBS, 339 glutamine, and 2-mercaptoethanol (ThermoFisher). Stimulated cells were surfaced stained for ILC2s and 340 per group. *P < .05, **P < .01, ***P < .001, unpaired t test. 581 experiments with 1-3 mice per group. *P < .05, **P < .01, ***P < .001, unpaired t test. 