Elevated CO2 Suppresses Specific Drosophila Innate Immune Responses and Resistance to Bacterial Infection

Elevated CO2 suppresses specific Drosophila innate immune responses and resistance to bacterial infection

Iiro Taneli Heleniusa,b,1, Thomas Krupinskia,1, Douglas W. Turnbullc, Yosef Gruenbaumd, Neal Silvermane, Eric A. Johnsonc, Peter H. S. Spornb,f, Jacob I. Sznajderb, and Greg J. Beitela,2

aDepartment of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, Evanston, IL 60208; bDivision of Pulmonary and Critical Care Medicine, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611; cInstitute of Molecular Biology, University of Oregon, Eugene, OR 97403; dDepartment of Genetics, Institute of Life Sciences, Hebrew University of Jerusalem, Jerusalem 91904, Israel; eDivision of Infectious Diseases, University of Massachusetts Medical School, Worcester, MA 01605; and fJesse Brown Veterans Affairs Medical Center, Chicago, IL 60612

Edited by Kathryn V. Anderson, Sloan–Kettering Institute, New York, NY, and approved September 4, 2009 (received for review June 2, 2009)

Elevated CO2 levels (hypercapnia) frequently occur in patients with (22, 23). In C. elegans,CO2 avoidance is mediated by specific obstructive pulmonary diseases and are associated with increased neurons (24, 25). The best characterized non-neuronal sensor of mortality. However, the effects of hypercapnia on non-neuronal CO2 to date is soluble adenylyl cyclase (26, 27, reviewed in ref. 28). tissues and the mechanisms that mediate these effects are largely However, soluble adenylyl cyclases have been lost in many evolu- unknown. Here, we develop Drosophila as a genetically tractable tionary lineages, including plants, yeast, Drosophila, and C. elegans model for defining non-neuronal CO2 responses and response path- (29). Therefore, responses to CO2 in these species must use other ways. We show that hypercapnia significantly impairs embryonic mechanisms. We sought to develop a genetically and molecularly morphogenesis, egg laying, and egg hatching even in mutants lacking tractable system for defining non-neuronal CO2 response pathways. the Gr63a neuronal CO2 sensor. Consistent with previous reports that We chose Drosophila because of extensive conservation of the hypercapnic acidosis can suppress mammalian NF-␬B-regulated in- hypoxia (30), nitric oxide (NO) (31), and nearly all other major nate immune genes, we find that in adult flies and the phagocytic signal transduction pathways. Further, Drosophila possesses a well- immune-responsive S2* cell line, hypercapnia suppresses induction of characterized, multicomponent innate immune system controlled IMMUNOLOGY specific antimicrobial peptides that are regulated by Relish, a con- by conserved signaling pathways that include NF-␬B-family tran- served Rel/NF-␬B family member. Correspondingly, modest hyper- scription factors (32, 33). The organism’s powerful in vivo and in capnia (7–13%) increases mortality of flies inoculated with E. faecalis, vitro genetics combined with the ability to rapidly assay immune A. tumefaciens,orS. aureus. During E. faecalis and A. tumefaciens and physiological responses has resulted in manifold contributions infection, increased bacterial loads were observed, indicating that to our understanding of mammalian innate immunity and immu- hypercapnia can decrease host resistance. Hypercapnic immune sup- nity in general. These include identification of the Toll family of pression is not mediated by acidosis, the olfactory CO2 receptor Gr63a, receptors (34) and RNAi as an antiviral mechanism (35, 36), or by nitric oxide signaling. Further, hypercapnia does not induce functioning of NF-␬B and Immune Deficiency (IMD)/TNF path- responses characteristic of hypoxia, oxidative stress, or heat shock. ways (32, 33, 37), defining roles of NO, Wnt, and insulin signaling Finally, proteolysis of the Relish I␬B-like domain is unaffected by in immunity (31, 38, 39), increasing understanding of host tolerance hypercapnia, indicating that immunosuppression acts downstream versus resistance in pathogenesis (40), and describing immunolog- of, or in parallel to, Relish proteolytic activation. Our results suggest ical roles of autophagy (41). Drosophila has been especially valuable that hypercapnic immune suppression is mediated by a conserved for investigating interactions between the immune system and response pathway, and illustrate a mechanism by which hypercapnia environmental factors, including endosymbionts, mating, circadian could contribute to worse outcomes of patients with advanced lung rhythm, feeding, and native microbiota (e.g., 42, 43). disease, who frequently suffer from both hypercapnia and respira- In this report we provide evidence that Drosophila melano- tory infections. gaster has specific physiological responses to hypercapnia. No- tably, hypercapnia suppresses expression of a subset of antimi- ␬ COPD ͉ hypercapnia ͉ Relish ͉ NF-␬B ͉ Gr63a crobial peptides regulated by highly conserved NF- B pathways, and there is a concomitant decrease in resistance of adult flies to specific bacterial pathogens. These results establish Drosoph- n average human produces 450 L of CO per day (1) and 2 ila as a general model for defining non-neuronal CO signaling elevated levels of CO (hypercapnia) in the pulmonary and/or 2 A 2 pathways and as a specific model for investigating suppression of circulatory system are associated with worse outcomes in patients innate immune responses by hypercapnia. with cystic fibrosis (CF) (2) and chronic obstructive pulmonary disease (COPD) (3, 4), currently the fourth leading cause of death Results in the US (5). In vitro and animal studies have shown that Hypercapnia Causes Specific Effects on Drosophila Physiology Inde- hypercapnia can suppress mammalian inflammatory responses ␬ pendent of Known Neuronal CO2-Sensing Pathways. To determine (6–8), including NF- B-regulated cytokine production (9–11), whether hypercapnia affects Drosophila physiology, we exposed which could contribute to the poor outcomes of patients with COPD and CF who frequently suffer from both hypercapnia and bacterial lung infections (12, 13). Hypercapnic immune suppression Author contributions: I.T.H., T.K., N.S., E.A.J., P.H.S.S., J.I.S., and G.J.B. designed research; may be evolutionarily conserved because hypercapnia, in combi- I.T.H., T.K., and D.W.T. performed research; I.T.H., T.K., D.W.T., Y.G., N.S., E.A.J., P.H.S.S., nation with hypoxia, also suppresses innate immune responses in J.I.S., and G.J.B. analyzed data; and I.T.H., T.K., and G.J.B. wrote the paper. shrimp, oysters, and crabs (14–16). However, the cellular pathways The authors declare no conflict of interest. that respond to CO2 and the physiological effects of hypercapnia This article is a PNAS Direct Submission. are poorly understood (reviewed in refs. 17–19). 1I.T.H. and T.K. contributed equally to this work CO2-sensing pathways in animals have been best defined in the 2To whom correspondence should be addressed. E-mail: [email protected]. nervous system (17, 20, 21). In Drosophila, the olfactory receptors This article contains supporting information online at www.pnas.org/cgi/content/full/ Gr63a and Gr21a are both required for avoidance behavior to CO2 0905925106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905925106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 24, 2021 Fig. 1. Hypercapnia affects Drosophila development and physiology independently of neuronal CO2 sensing. (A–C) Drosophila embryonic development is disrupted by hypercapnia as revealed by luminal staining of the embryonic tracheal (airway) system whose morphogenesis requires interaction with many distinct tissues. Culturing wild-type embryos in 13% CO2 causes moderate defects in 20% of embryos (B, n ϭ 111), while 19.5% CO2 causes severe defects in 28% of embryos (C, n ϭ 71) and increases the prevalence of moderate defects to 41%. Moderate defects (B) include breaks in the main airways (arrowheads) and missing or ectopic interconnections of dorsal branches (arrows). Severely abnormal tracheal development reveals gross embryonic patterning and/or morphogenesis defects (C). Representative images for each phenotype are shown. (D and E) Hypercapnia (24 h, D) decreases hatching of eggs laid in normocapnia by wild-type (WT) or mutant ﬂies homozygous for a null allele in the neuronal CO2 receptor, Gr63a. In 13% CO2, over 90% of WT embryos hatch after 48 h (E), but in 19.5% CO2 only approximately 30% hatch. (F) Hypercapnia reduces the number of eggs laid in 48 h by WT and Gr63a-null females mated in normocapnia. (G) As in mammalian cells (69), hypercapnia (1 h) causes endocytosis of the Na,K-ATPase in S2 cells. (H) Suppression of the antimicrobial peptide (AMP) Diptericin by hypercapnia in S2* cells is concentration- dependent. (Protein levels of AMPs were not assessed because antibodies against Drosophila AMPs are not available.) *, P Ͻ 0.05; **, P Ͻ 0.005.

flies to 13% CO2 (PCO2 94 mm Hg) and 19.5% CO2 (PCO2 140 mm 13% CO2 for 24 h causes fewer than 500 genes to be up-regulated Hg), while maintaining O2 at 21% (see SI Text). These CO2 levels or down-regulated Ͼ1.5-fold, and fewer than 10 by Ͼ10-fold. are well below the Ͼ35% concentration at which CO2 becomes Importantly, the regulated genes define discrete physiological func- anesthetic (44). Hypercapnia causes concentration-dependent de- tions. All of the up-regulated gene ontology (GO) families have GO fects in embryonic development. Thirteen percent CO2 results in annotations relating to metabolic functions (Fig. S1A), and 67% of 20% of embryos having moderate defects such as malformations of the down-regulated gene ontology families have either immune- or the airway system (compare Fig. 1 A and B) and significantly slows fertility-related GO annotations (Fig. S1B). The strong down- hatching of eggs laid by normocapnic mothers (Fig. 1 D and E). At regulation of the chorion and vitelline membrane genes required 19.5% CO2, there is severe disruption of embryonic development for egg production (Fig. S2A) is consistent with the dramatic with approximately 30% of exposed embryos showing large-scale decrease in fecundity of flies in hypercapnia. Critically, CO2 does patterning and morphogenesis defects (Fig. 1C), and Ͼ70% of eggs not induce genes characteristic of responses to hypoxia, heat shock, failing to hatch (Fig. 1E). The life span of adult flies is not affected or oxidative stress (Fig. S2 B–D), indicating that the responses to elevated CO2 are mediated by distinct pathways. Together, these by 13% CO2 (Fig. 3A), but hypercapnia does cause a concentration- dependent reduction in the number of eggs that females lay (Fig. results show that in Drosophila, hypercapnia does not cause global changes in gene expression, but instead has a very specific tran- 1F). These effects of CO2 on development and fertility are consistent with those we recently reported in C. elegans (45), with the scriptional signature. exception that 19% CO increases worm life span. Importantly, flies 2 Hypercapnia Suppresses Select Antimicrobial Peptide Genes. The homozygous for a null mutation in the neuronal CO receptor 2 microarray data show that the down-regulated genes with immune- Gr63a (22, 23) are as sensitive to hypercapnia as wild-type flies in related GO annotations that are enriched in elevated CO condi- the egg hatching and laying assays (Fig. 1 D–F). Thus, many 2 tions are antimicrobial peptides (AMPs), which are important physiological effects of hypercapnia are mediated by an as-yet effectors of the Drosophila innate immune system (Fig. 2A). This uncharacterized CO response pathway(s). 2 observation parallels previous reports indicating that hypercapnic We next asked whether Drosophila shares with mammals any acidosis can suppress innate immune responses in mammals (6–11, specific response to hypercapnia. We previously reported that in 48, 49). Given the prevalence of pulmonary infections in CF and human alveolar epithelial cells, hypercapnia causes endocytosis of COPD patients (12, 13) and that hypercapnia is a risk factor for the Na,K-ATPase (46). Analysis of surface abundance of Na,K- mortality in both of these debilitating diseases (2–4), we focused on ATPase in Drosophila S2 cells reveals that hypercapnia also causes defining the effects of elevated CO2 on the fly innate immune concentration-dependent endocytosis of the sodium pump in S2 system. We confirmed and extended the microarray results using cells (Fig. 1G). This result supports the existence of cell- quantitative real-time reverse transcriptase PCR (qPCR) and autonomous CO2 responses that do not depend on the neuronal found that hypercapnia can indeed suppress expression of AMPs in CO2-sensing pathways. That both Drosophila and human cells adult flies (Fig. 2B). endocytose their Na,K-ATPase in response to hypercapnia suggests To identify a simplified in vitro system for investigating hyper- that some CO2 responses are conserved between mammals capnic immune suppression, we tested the effects of hypercapnia on and flies. the expression and induction of AMPs in Drosophila S2* cells, a phagocytic immune-responsive cell line whose viability we deter- Hypercapnia Causes Specific Effects on Gene Expression. To investi- mined to be unaffected by 13% CO2 (Fig. S3A). In flies, AMPs are gate the molecular basis of the physiological effects of hypercapnia regulated by one or both of the well characterized and conserved and identify CO2-responsive promoters to use as markers for Toll and TNF-like IMD pathways (32, 50). Since S2* cells in culture dissecting CO2-signaling pathways, we performed microarray anal- lack the extracellular components required to activate the Toll ysis on adult flies. Similar to the limited changes in gene expression receptor, E. coli peptidoglycan (PGN) induces the IMD pathway. seen in neonatal mice raised in CO2 (47), exposure of adult flies to However, there is likely to be some cross-talk to intracellular parts

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905925106 Helenius et al. Downloaded by guest on September 24, 2021 Fig. 2. Hypercapnia down-regulates specific antimicrobial peptides. (A and B) Hypercapnia (24 h) suppresses specific AMPs in vivo in unchallenged adults as shown by microarray analysis (A) and confirmed by quantitative PCR (B). (C and D) Hypercapnia suppresses specific AMPs in vitro (10 h CO2 for C and D; PGN challenge at5hinD). *, P Ͻ 0.05; **, P Ͻ 0.005. PGN ϭ E. coli peptidoglycan; Drs ϭ Drosocin; Att ϭ Attacin; Dpt ϭ Diptericin; Cec ϭ Cecropin; Def ϭ Defensin; Drm ϭ Drosomycin; Mtk ϭ Metchnikowin.

of the Toll pathway as well (51, 52). PGN challenge of S2* cells modest 5% CO2 causes a 50% suppression of Dpt induction (Fig. induces expression of all AMPs in air and CO2 (Fig. 2 and Fig. S4D), 1H), S2* cells provide a powerful in vitro tool for elucidating the but in hypercapnia the induced levels of some AMPs are suppressed molecular details of CO2-response pathways. (Fig. 2 C and D and Fig. S4D). Diptericin (Dpt), Attacin, and Drosomycin are consistently suppressed 3- to 5-fold, whereas Hypercapnia Increases Mortality to Specific Bacterial Infections. An- Metchnikowin is not (Fig. 2 C and D). Importantly, hypercapnia timicrobial peptides are a crucial element in the fly’s defense against also differentially suppresses AMP expression in whole flies (Fig. 2 pathogens (53). We therefore asked whether hypercapnia reduces A and B), and the responses in S2* cells reasonably approximate the ability of flies to survive infection with bacteria, including the those observed in vivo. Further, as treating S2* cells with even a natural Drosophila pathogen E. faecalis (54), the human pathogen IMMUNOLOGY

Fig. 3. Hypercapnia decreases resistance of Drosophila to specific bacterial infections. (A) Hypercapnia does not affect Drosophila life span. (B–H) Hypercapnia slightly increases death of flies inoculated with sterile PBS (B) or with E. coli (E), but significantly increases mortality at CO2 levels as low as 7% after inoculation with A. tumefaciens (C), the human pathogen S. aureus (D), and the Drosophila natural pathogen E. faecalis (F). Immune suppression does not require the neuronal CO2 receptor Gr63a (G). (H) Pretreatment of flies with 9% CO2 before S. aureus infection in air is sufficient to increase mortality, even when flies are cultured in air after inoculation. For A–H, unless otherwise noted, flies were exposed to indicated CO2 level for 24 h before inoculation and returned to hypercapnia until end of assay. We show representative results for the lowest CO2 levels at which significant effects on mortality were consistently observed. All experiments were done in triplicate and the trial with the middle P value is shown. (I–L) Hypercapnia increases the bacterial load for strains causing increased mortality during hypercapnia. Horizontal lines show medians. Calculation of P values described in SI Text. CFU, colony forming units. (M–P) Effects of hypercapnia on bacterial growth. Note that S. aureus growth is dramatically reduced in 7% CO2 even though hypercapnia increases mortality of flies infected with S. aureus. Error bars smaller than the data-point symbols are not shown.

Helenius et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 24, 2021 S. aureus, which is commonly isolated from COPD and CF patients (13, 55), and A. tumefaciens, which has previously been reported to increase mortality in flies lacking a conserved component of NF-␬B immune pathways (56). Elevated CO2 slightly decreases survival of flies inoculated with sterile PBS (Fig. 3B)orwithE. coli (Fig. 3E), which is not considered a Drosophila pathogen (53). Wounding modestly reduces average life span (compare Fig. 3 A and B air conditions), but hypercapnia alone does not (Fig. 3A). Therefore, the marginally decreased survival in elevated CO2 after non- pathogenic inoculations suggests that hypercapnia could interfere with wound-healing, which is closely linked to AMP expression (57). Survival of flies infected with E. faecalis (Fig. 3F) was significantly decreased by moderately elevated CO2 (13% CO2). Strikingly, CO2 levels as low as 7% increase mortality to A. tumefaciens and S. aureus (Fig. 3 C and D). Furthermore, elevated CO2 increases mortality to A. tumefaciens in flies lacking Gr63a (Fig. 3G), indicating that hypercapnic immune suppression is mediated by mechanisms independent of known neuronal CO2- sensing pathways. A critical issue is whether decreased survival of infected flies results from effects of hypercapnia on the host or the pathogen. To Fig. 4. Hypercapnia suppresses immune responses independent of pH, NO, test for host effects, we exposed flies to 9% CO2 for 24 h and returned them to normocapnia after inoculation with S. aureus. and proteolytic activation of Rel. (A) Suppression of Diptericin (Dpt) in PGN- challenged S2* cells is not attenuated or enhanced by the NO synthase Hypercapnic pretreatment of the host significantly increases mor- inhibitor L-NAME (1 mM) or the NO donor SNAP (1 mM). (B) Hypercapnia tality after infection, although the pathogen was never exposed to suppresses Dpt induction 5-fold when the culture medium is adjusted to pH7.0 elevated CO2 (Fig. 3H). Thus, hypercapnia compromises the ability with NaOH, but Dpt induction is only suppressed approximately 2-fold by of flies to combat bacterial infections independently of effects of Mops at pH6.5 and by NaCl and Hepes at pH7.0. (C) Hypercapnia does not alter hypercapnia on the pathogen. Relish (Rel) cleavage in response to PGN challenge. GFP immunoblot of We also investigated whether hypercapnia affects pathogens whole-cell lysates of S2 cells expressing GFP-Rel. (D and E) Models for hypercapnic suppression of innate immune effectors (see text for discussion). , P Ͻ by measuring their growth rates in LB media at elevated CO2 * Ͻ levels. Growth of E. coli and E. faecalis in media is not signifi- 0.05; **, P 0.005 between CO2 condition and equivalently treated air condition (A) or untreated air condition (B). cantly affected by elevated CO2 (Fig. 3 M and N). However, despite the increased mortality of flies infected with A. tumefaciens and S. aureus, growth of these bacteria is actually markedly tested possible roles of candidate signaling pathways in hypercapnic reduced by hypercapnia (Fig. 3 O and P). Because decreased immune suppression. Nitric oxide (NO) has important roles in bacterial growth would be expected to improve fly survival, the innate immune function in Drosophila and mammals (31, 58), and observed increase in mortality underscores the deleterious ef- hypercapnia has been proposed to modulate NO-dependent path- fects of hypercapnia on Drosophila host defenses. ways and inflammatory oxidants (59). However, hypercapnic suppression of Dpt in S2* cells is unaffected by either a NO synthase Hypercapnia Decreases the Resistance to Bacterial Infections. The inhibitor or a NO donor (Fig. 4A), indicating that hypercapnic survival of a host during infection is determined by its ability to immune suppression is not mediated via signaling by NO. resist the growth of pathogens and its ability to tolerate the presence Another candidate mediator of hypercapnic immune suppres- of an infection. To distinguish whether resistance or tolerance is sion is acidosis, which is known to regulate immune responses affected by hypercapnia, we determined bacterial loads in flies [reviewed in (60, 61)]. CO2 reacts with water to form carbonic acid infected with E. coli, E. faecalis, and A. tumefaciens (see SI Text). (H2CO3) and previous investigations of hypercapnic innate immune Hypercapnia increases bacterial loads in WT and Gr63a mutant suppression in mammalian immune cells have implicated—but not flies infected with strains that increase mortality (Fig. 3 J–L). Thus, proven—acidosis as the critical mediator (9, 11, 62). Here, we show hypercapnia reduces resistance to some bacterial infections. that elevated CO2 levels can suppress immune responses in S2* cells These results are consistent with hypercapnic acidosis increasing independent of extracellular pH effects. Acidifying S2* cell culture lung bacterial load in a rat pneumonia model (8), and with media with 19 mM Mops in normocapnia causes only a 2-fold hypercapnic hypoxia increasing bacterial load in infected marine suppression of Dpt expression, less than half that caused by 13% invertebrates (15). CO2, which is associated with an equivalent decline in pH (Fig. 4B). Notably, 25 mM NaCl or Hepes at neutral pH causes suppression Hypercapnia Rapidly Suppresses Innate Immune Responses in Vitro. equal to that of acidosis, making it difficult to discern acidotic from To further investigate the nature of hypercapnic immune sup- ionic or non-specific effects. We definitively demonstrate effects of pression, we determined how hypercapnia affects the kinetics of CO2 independent of extracellular acidosis by maintaining the S2* innate immune responses in S2* cells. Hypercapnia suppresses cell media at neutral pH during exposure to 13% CO2 and finding rather than delays innate immune responses of PGN-challenged that hypercapnia suppresses Dpt induction to the same extent seen S2* cells, as the magnitude of the responses at all times is in hypercapnic acidosis (Fig. 4B, black bars). Thus, in Drosophila reduced (Fig. S4A). This suppression exhibits rapid onset (Fig. cells, hypercapnia causes a consistent pattern of immune suppres- S4B) and recovery (Fig. S4C). sion distinct from the effects of extracellular acidosis. Importantly, this result parallels our results with mammalian alveolar macro- Hypercapnic Immunosuppression Is Not Mediated by NO or Acidosis. phages that demonstrated that CO2 suppresses expression of par- As discussed above, the effects of hypercapnia on Drosophila ticular NF-␬B regulated cytokines independent of acidosis (48). physiology and the immune responses of S2* cells are not mediated via neuronal CO2 sensing, and hypercapnia acts via pathways Hypercapnia Acts Downstream of, or in Parallel to, Relish Activation. distinct from hypoxia, heat shock, and oxidative stress. Because How does hypercapnia modulate innate immune responses? We non-neuronal CO2-sensing pathways have not been defined, we used cultured S2 cells to investigate how hypercapnia suppresses

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0905925106 Helenius et al. Downloaded by guest on September 24, 2021 AMPs by examining PGN-induced activation of Relish (Rel), a of metabolic activity, with elevated CO2 indicating excessive met- homolog of the mammalian Rel/NF-␬B transcription factors. Rel abolic load. We have shown that hypercapnia suppresses select acts in the IMD pathway and contains an N-terminal Rel physiological functions that are known to be metabolically demand- homology domain (RHD) that is endoproteolytically cleaved ing, including immune responses (65), egg-laying, and Na,K- from a C-terminal inhibitory I␬B-like region in response to PGN ATPase activity, which consumes up to 40% of the energy supply (63). The RHD then binds ␬B-sites on AMP promoters and in some cell types (66). This may allow for the reallocation of energy induces their expression (32). In S2 cells exposed to 13% to more immediate needs such as powering the flight muscles. hypercapnia, there is no reduction in the PGN-induced proteo- Consistent with this hypothesis, our microarray data reveal that all lytic cleavage of Rel (Fig. 4C). Therefore, hypercapnia inhibits of the Drosophila gene ontology families up-regulated by hyper- expression of Rel targets downstream of, or in parallel to, capnia are involved in metabolism (Fig. S1A) and there is com- proteolytic activation of Rel. Together with work in human and monality between the genes regulated during hypercapnia and mouse macrophages showing that hypercapnia suppresses starvation (Fig. S7). Furthermore, our previous work has shown ␬ NF- B targets independent of acidosis and without blocking that in mammalian cells, hypercapnia activates AMP-activated ␬ ␣ degradation of I B (48), our results support the idea of a protein kinase (AMPK), a central regulator of metabolism (46). conserved mechanism of hypercapnic immune suppression. AMPK is activated by conditions that deplete ATP reserves, such Discussion as hypoxia or rapid muscle contraction. We suggest that in some tissues, CO could act as a diffusible signal that regulates metab- We have systematically investigated mechanisms that could 2 olism. A metabolically active tissue would produce CO that could mediate responses to elevated CO . The effects of hypercapnia 2 2 decrease activity of a responding tissue to preserve energy resources are present in flies lacking the Gr63a gustatory CO receptor and 2 for the source tissue. are therefore mediated by mechanisms that are distinct from Hypercapnic suppression of innate immune and inflammatory known neuronal CO2-sensing pathways, and that can act cell autonomously. These effects are not simply a consequence of responses has important implications for human health. Previous cellular damage because hypercapnia does not induce changes in reports have suggested that hypercapnia can have beneficial gene expression characteristic of other environmental insults effects when ‘‘permissive hypercapnia’’ regimens are used during such as hypoxia, oxidative stress, or heat shock, nor does mechanical ventilation (reviewed in ref. 67). Reduced inflam- hypercapnia decrease fly life span or cell viability. mation is also observed when CO2 is used as an insufflatant

In Drosophila, the majority of gene expression changes in during laparoscopic surgery (68). In these situations, which do IMMUNOLOGY hypercapnia are accounted for by increased expression of metabolic not involve defenses against pathogens, hypercapnic suppression genes and by down-regulation of reproductive and immune genes. of inflammatory responses may reduce tissue damage and This specific profile of changes is distinguishable from those of improve outcomes. However, when pathogens are present, as in other immune-related insults such as Mycobacterium infection, patients with COPD or cystic fibrosis, immune suppression by which down-regulates metabolic genes and up-regulates the innate hypercapnia could increase susceptibility to, or exacerbate con- immune response (Fig. S5), and from Pseudomonas infection, which sequences of, bacterial infections. Experimental support of this appears to suppress AMPs differently than CO2 (Fig. S6). Inter- possibility is provided by O’Croinin et al. (8) who recently estingly, despite some distinctions, many metabolic and other genes showed that in a bacterial pneumonia model, rats exposed to 5% regulated by starvation are similarly regulated by hypercapnia (Fig. CO2 had higher lung bacterial counts and more structural S7). The effects of CO2 on Drosophila innate immunity are most damage than normocapnic controls. Together, the Drosophila likely not due to decreased feeding, since we find a similar pattern and rat results suggest that hypercapnia is not simply a marker of AMP down-regulation in S2* cells as in adult flies. We conclude of disease status, but rather that it can actively contribute to the that the gene expression changes we observe are specific and unique progression of infection. The current lack of understanding of to hypercapnia. the molecular pathways that mediate CO2 responses severely To begin dissecting the mechanisms of immune gene regulation limits our ability to assess and intervene in pathological situa- by hypercapnia, we have investigated its effects on the TNF-like tions involving hypercapnia. Our findings establish Drosophila as IMD/Rel pathway. Our results show that hypercapnia inhibits a genetically and molecularly tractable system in which to define expression of Rel targets downstream of, or in parallel to, proteo- non-neuronal CO response pathways that modulate host de- ␬ ␣ 2 lytic cleavage of the I B -like domain of Rel. This result parallels fenses and other physiologically important processes. the findings of Wang et al. (48) who show in mammalian macrophages that hypercapnia suppress NF-␬B-regulated genes without Materials and Methods ␬ ␣ affecting proteolysis of I B . We propose two models for how a See SI Text for additional detail. CO2 response pathway might suppress transcription of Drosophila CO2 exposures performed in BioSpherix C-Chambers (BioSpherix Ltd) fitted Rel targets such as Dpt. In the first model, a CO2 response pathway with a ProCO2 regulator supplied with 20% CO2/21% O2/59% N2. Unless other- may negatively regulate one of the components of the Rel tran- wise noted, 5 h before PGN challenge, cells were resuspended in pre-equilibrated scription complex or block Rel nuclear import (Fig. 4D). Alterna- media. tively, and analogous to the regulation of hypoxia-responsive genes ␮ by the HIF transcription factor (reviewed in ref. 64), a CO2- Cell Culture. Cells were primed with 1 M ecdysone 15 h before CO2 treatment and challenged with 100 ng/mL PGN for 5 h (Fig. 2D) or 2.5 h (Figs. 1H and 4 A and response pathway would activate a hypothetical CO2-responsive B, and (Fig. S4 B and C). Surface biotinylation was performed as previously factor (CO2RF) that binds specific CO2-response elements described (69). L-NAME (1 mM) or 1 mM SNAP was added 10 h or 3 h, respectively, (CO2RE) and then inhibit the Rel complex or directly suppress Dpt expression (Fig. 4E). The conceptual difference between the two before CO2 treatment as described in ref. 70. Media pH was adjusted by adding models is that, in the first, genetic removal of the component of the reagents to the indicated final concentrations: 19 mM Mops, 25 mM NaCl, 12.5 mM Hepes, and 25 mM NaOH (pre-equilibrated in CO2 overnight). Rel complex targeted by the CO2-response pathway would signif- Bacterial infection and CFU counts were performed as described previously icantly alter Dpt transcription even in the absence of CO2.In (71, 72). Kaplan–Meier survival plots and P values generated using GraphPad contrast, removal of the CO2-responsive factor in the second model Prism software from combined triplicates for a total of approximately 75 flies would render the promoter unresponsive to CO2 regulation, with- per survival curve. Inocula CFUs shown in Fig. S8. out affecting basal or induced transcription of Dpt in normocapnia. Why should elevated CO2 levels cause the physiological re- Developmental and Fecundity Assays. Developmental defects were assessed in sponses we observe? One possibility is that CO2 serves as a read-out embryos exposed to elevated CO2 for 19 h. For the egg hatching assay, eggs laid

Helenius et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 24, 2021 by flies in air were collected for 1–2 h, counted, transferred to CO2 conditions, and ACKNOWLEDGMENTS. We thank M. Ducommum and L. Welch for techni- scored at 24 h or 48 h. Fecundity was determined as described in ref. 73. cal assistance; A. Hauser, E. Lecuona, P. O’Farrell, D. Schneider, E. Nester, and L. Vosshall for fly strains, reagents or advice; and two anony- Whole Fly Microarrays. Triplicates of at least 30 5-day-old male and female flies mous reviewers for constructive suggestions. This work was funded by American Heart Association Grant-in-aid AHA0855686G (to G.J.B.) and a were used as described in ref. 74. National Center for Biotechnology Infor- predoctoral fellowship AHA0715562Z (to I.T.H.); National Institutes of mation GEO accession number is GSE17444. Health Grants R01GM069540 (to G.J.B.), NIH-R01HL48129 (to J.I.S.), NIH- R01HL85534 (to J.I.S. and Y.G.), NIH-R01AI1060025 (to N.S.), NIH- Statistical Analyses. Two-tailed Student’s t tests were used unless noted. Error R01HL072891 (to P.H.S.S.); Veteran’s Administration Merit Review bars indicate standard deviation. Air vs. CO2 CFUs, t tests were performed on (P.H.S.S.); and American Chemical Society Research Scholar Grant RSG-03– the natural logarithms of CFU counts as described in ref. 75. 154-01-DDC (to E.A.J.).

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