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Mycobacterium tuberculosis H37Rv Induces Expression of PDE4A and PDE7A in Human Macrophages

Adrian Naoun1,2, Chrissy M Leopold mediator derives from its ability to suppress innate Wager1, Eusondia Arnett1, and Larry S immunity functions in macrophages, monocytes, Schlesinger1 and neutrophils by limiting pro-inflammatory cytokine release. Despite its known effects, the 1Texas Biomedical Research mechanisms underlying cAMP activation in Institute, San Antonio, Texas, response to M.tb are incompletely understood, United States of America. 2The particularly in human macrophages. Preliminary University of Texas at San Antonio, data indicate that cAMP levels are increased in human monocyte-derived macrophages (hMDMs) San Antonio, Texas, United States following infection with virulent M.tb H37Rv and of America. attenuated M.tb H37Ra. (PDEs) comprise a group of that degrade cAMP to regulate signal transduction. We hypothesize that elevated cAMP levels induce Abstract of certain PDEs as a host response mechanism to The World Health Organization reported that one- degrade M.tb-induced cAMP. Gene expression fourth of the global population is infected studies demonstrated that transcription of PDE4A with Mycobacterium tuberculosis (M.tb), the and PDE7A increased 48 and 72 h after infection, causative agent of an airborne infectious disease whereas PDE3A and PDE5A remained unaltered. known as tuberculosis (TB). In 2017, TB alone These data suggest that human macrophages up- caused 1.6 million deaths. M.tb is an intracellular regulate PDE expression to limit M.tb from pathogen equipped with specialized evolutionary dampening the immune response via high cAMP traits to evade immune mechanisms. Upon levels. Further studies will demonstrate the clinical inhalation, macrophages phagocytose M.tb and feasibility of cAMP degradation as a novel host- become a niche due to their inability to resolve the directed therapy to reduce M.tb pathogenesis. infection. The intracellular growth of M.tb is influenced, in part, by host transcription factors and Host-directed therapy, immunosuppressive second messengers like cyclic Keywords: adenosine monophosphate (cAMP). The macrophages, Mycobacterium tuberculosis, importance of cAMP as an inflammatory response pathogenesis, signal transduction, cAMP, PDE, gene regulation.

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1. Introduction Mycobacterium tuberculosis is Alveolar macrophages (AMs) are primarily a pulmonary pathogen tissue-resident lung phagocytes transmitted airborne through droplet responsible for maintaining lung nuclei from active TB patients. The homeostasis, clearing cellular debris, available two-phased chemotherapy and defending against pathogenic regimen has failed to eradicate TB due invasion [7]. AMs phagocytose M.tb to the intrinsic obstacles related to after the intracellular pathogen has sustaining treatment for at least six traversed several infection points along consecutive months [1]. The emergence the respiratory tract. However, the vast of multidrug-resistant (MDR) and repertoire of AMs is insufficient to extensively drug-resistant (XDR) strains resolve the infection. M.tb manipulates poses a threat to humanity as core a myriad of processes in a multipronged treatments have been rendered effort to establish a permissive inefficacious. The therapeutic approach environment and subvert the host for drug-resistant TB is cumbersome immune response. As for evasion due to less-efficient, expensive, and mechanisms, M. tuberculosis can inhibit toxic substitutes to the first-line agents phagolysosomal maturation, manipulate [2]. Adjunctive host-directed therapies host cellular trafficking, neutralize the (HDTs) have emerged as novel host’s oxidative bursts, as well as alternatives to attenuate M.tb inhibit apoptosis and autophagy pathogenesis. The principle of HDTs is pathways [8-10]. M.tb to augment the host’s immune immunopathology is also influenced, in mechanisms and limit excessive tissue part, by eicosanoid signaling pathways, pathology; therefore, decreasing transcription factors, and treatment duration while reducing immunosuppressive second messengers morbidity and mortality [3]. M.tb can like cyclic adenosine monophosphate affect all compartments of the (cAMP). respiratory tract (i.e., nose, sinuses, pharynx, larynx, trachea, bronchi, Cyclic AMP remains the archetypal bronchioles, and lungs); however, it second messenger due to its ability to remains unclear if the disease regulate a wide range of essential manifestations arise due to primary cellular events such as gene expression, infection or reactivation either locally or cell division, metabolism, phagocytosis, in secondary tissues [4]. The and the immune response [11]. The Mycobacterium bovis Bacillus signal transduction pathway initiates Calmette-Guérin (BCG) is a live when an extracellular messenger (i.e., attenuated vaccine, which has been neurotransmitter, chemokine, lipid effective in mitigating severe mediator, hormone, or drug) binds to disseminated forms of TB in children the G protein-coupled receptor (GPCR), but remains unsuccessful in diminishing which is architecturally composed of primary infections or the reactivation of seven transmembrane helices [12]. latent TB [5]. There are no Ligand binding to the GPCR promotes environmental reservoirs for M. the exchange of GDP/GTP in the G tuberculosis; consequently, it acts as protein α subunit (Gαs) to facilitate the both a pathogen and a symbiont in βγ complex dissociation [13]. The free humans [6]. It is crucial to elucidate the Gαs stimulates the adenylyl host-pathogen interactions to aid in the cyclase (AC) to catalyze the cyclization eradication of TB. of ATP and yield cAMP, as well as

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pyrophosphate [14]. Elevations in pathways, as well as physiological intracellular cAMP suppress the innate processes [21]. There are 11 gene- immunity functions of macrophages, related families named PDE1 to PDE11 which account for 95% of the [14]. Although the members of each leukocytes present in the lower PDE family display a considerable respiratory tract under steady-state divergence of amino acid sequences, the conditions [15]. functional relationship remains the same according to their substrate, In the AMs, cAMP exerts inhibitory inhibitor specificities, and enzymatic effects by modulating the production of activity regulation [22]. PDEs hold cytokines, chemokines, and lipids [16]. promising therapeutic potential due to Additionally, a variety of cell activation their unique tissue distribution and gene components such as phagocytosis, expression patterns. For instance, PDE5 reactive oxygen intermediate (ROI) inhibitors are available for treating male generation, and the production of erectile dysfunction and pulmonary inflammatory mediators can be hypertension; in contrast, PDE3 inhibited [17]. For instance, the pro- inhibitors target short-term dilated inflammatory cytokines TNF-α, MIP- cardiomyopathies [23,24]. Adjunct 1α, and leukotriene B4 are inhibited by HDTs such as the pharmacological cAMP-dependent pathways; in contrast, inhibition of PDE4 augment the the production of the anti-inflammatory bactericidal activity of isoniazid while cytokine IL-10 and the suppressor of reducing pulmonary fibrosis in M.tb- cytokine signaling-3 (SOCS3) is infected mouse and rabbit models enhanced [18]. The role of downstream [25,26]. The regulation of anti- and pro- cAMP-activated effector complexes inflammatory cytokines can be such as Protein Kinase A (PKA) and translated into a marked reduction of Epac1 remains poorly understood. The M.tb’s granulomatous pathology, which virulence arsenal of M.tb can is associated with clinical persistence. manipulate the cAMP-PKA system to The success of innovative HDTs has inhibit the phagosomal assembly of extrapolated the interest in PDEs to actin; thus, promoting its intracellular various research disciplines. survival [19]. The secretion of interferon-gamma (IFN-γ) by type 1 T- In the present study, we employed the helper cells (Th1) activates human monocyte-derived macrophage macrophages in cell-mediated immunity (hMDM) model to determine cAMP during M.tb infection. However, fluctuations and the gene expression patients with advanced TB disease signature of PDEs in response to M.tb exhibit an inhibition of IFN-γ infection. We preliminarily discovered production due to the enzymatic activity that cAMP levels are increased in of type I PKA [20]. hMDMs following infection with attenuated M.tb H37Ra and virulent M.tb The delicate intracellular balance of H37Rv. Gene expression studies via cAMP is alternatively regulated by a qRT-PCR demonstrated that family of enzymes known as cyclic transcription of PDE4A and PDE7A is nucleotide phosphodiesterases (PDEs). induced by the M.tb strain H37Rv in PDEs hydrolyze cyclic nucleotide hMDMs. In contrast, the gene second messengers such as cAMP and expression of PDE3A and PDE5A is cyclic guanosine monophosphate not significantly altered. Thus, our (cGMP) to regulate signal transduction research identifies new potential targets

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to modulate M.tb-induced cAMP in Texas Biomedical Research Institute macrophages, and further investigation following approved facility and safety is required to determine the feasibility guidelines. of cAMP regulation as a novel HDT. 2.3 M.tb infection of hMDMs Single cell suspensions of M.tb in RHH 2. Materials and Methods (0.1% human serum albumin [CSL Behring, King of Prussia, PA], RPMI, 2.1 Monocyte isolation and culture of and 10mM HEPES [Life human monocyte-derived Technologies]) were added to the macrophages (hMDMs) hMDMs at a Multiplicity of Infection The MDM preparation process is (MOI) 5 or the equivalent of 5 bacteria described elsewhere [27,28]. Briefly, per macrophage. The cells were peripheral blood samples were collected incubated for 2 h at 37°C to facilitate from healthy donors following bacterial phagocytosis, with the first 30 approved protocols from the min on a platform shaker [31]. hMDMs Institutional Review Board and Texas were then washed to remove Biomedical Research Institute. All extracellular bacteria and incubated in donors were informed and provided RPMI with 2% autologous serum. written consent. Peripheral blood hMDM monolayer integrity was mononuclear cells (PBMCs: 80% assessed during the indicated time monocytes and 20% lymphocytes) were points by cell viability. Pictures were isolated from heparinized blood with captured with 10X and 40X objective Ficoll-Paque separation media (GE lenses (Olympus CKX41). Healthcare, Uppsala, Sweden). The 2.4 RNA isolation and quantification PBMCs were cultured with RPMI hMDMs in triplicate wells were lysed (Invitrogen, Carlsbad, CA) as well as with TRIzol (Invitrogen) according to 20% autologous serum in Teflon wells the manufacturer’s recommendations. (Savillex, Eden Prairie, MN) for five The RNA samples were centrifuged at days at 37 ˚C in 5% CO2. PBMCs were 4˚C for 15 min under a g-force of harvested and transferred to tissue 14,000 after the addition of chloroform culture plates supplemented with RPMI (0.2 mL). The upper aqueous phase was and 10% autologous serum. After 2 h, isolated, and 500 µL of isopropanol lymphocytes were removed by washing, added. The samples were centrifuged at while hMDMs adhered to the plates to 14,000 x g for 10 min. The pellet was form a monolayer. The hMDMs were washed with 500 µL of 75% ethanol incubated overnight prior to infection. and then centrifuged for 5 min at 14,000 2.2 Bacterial strains x g. The precipitate was dissolved in 35 The lyophilized M.tb strain H37Rv was µL of Ultrapure distilled water obtained from the American Tissue (Invitrogen). Confirmatory readings Culture Collection (ATCC, Manassas, were conducted with the NanoDrop VA). Single cell suspensions were 1000 (ThermoFisher Scientific) to prepared as described previously quantify the RNA and assess its purity. [29,30]. The Petroff-Hausser Chamber 2.5 Gene expression studies via qRT- aided in the determination of clumping PCR degree (<10%) as well as bacterial The RNA samples were reverse concentration. The CFU assays transcribed into cDNA with SuperScript confirmed bacterial viability (≥90%). III Reverse Transcriptase (Invitrogen), M.tb infections of hMDMs were dNTPs, and random primers following conducted in the BSL3 laboratory of the the manufacturer’s guidelines. Gene

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expression was determined by results are reported as mean ± SD with quantitative real-time PCR (qRT-PCR) P ˂ 0.05 considered significant. All data via TaqMan Gene Expression Assays were analyzed and plotted using (Applied Biosystems, Foster City, CA) GraphPad Prism 8. and a 7500 Real-Time Thermocycler (Applied Biosystems). The gene 3. Results expression values were analyzed by the ΔΔCt method, in which β-actin served M.tb strains H37Ra and H37Rv induce as a housekeeping gene for cAMP expression in hMDMs normalization controls. Cyclic nucleotide second messengers 2.6 cAMP ELISA partly mediate the immunopathology of Supernatants were collected from M.tb- hMDMs during M.tb infection. M.tb infected hMDMs at the indicated time induces an acute increase in cAMP points, and cAMP was measured by intramacrophage concentrations as a competitive binding ELISA (Cayman virulence factor to inhibit inflammatory Chemical, Ann Arbor, MI) according to mediators such as cytokines, the manufacturer’s recommendations. chemokines, and lipids [16]. We 2.7 Statistical Analysis hypothesized that elevated cAMP levels PDE gene expression results are induce the gene expression of certain presented as the fold change ± SEM PDEs as a host response mechanism to unless otherwise indicated in the figure restore cellular homeostasis. We legends. The trend remained constant infected hMDMs with the M.tb strain for each donor; however, biological H37Rv at MOI 5. This multiplicity ratio replicates displayed a distinct ensures that approximately 30% of magnitude of change. Therefore, the MDMs will be infected with the data were normalized to an internal bacteria. Figure 1 shows the intact control. A one-way ANOVA was MDM monolayer in uninfected (1A) performed, followed by Dunnett’s and infected (1B) cells at 48 h post- multiple comparisons test. The cAMP infection.

Figure 1. M.tb infection of hMDMs. are unclear in M.tb-infected hMDMs. Uninfected control (A) compared to Therefore, we measured cAMP hMDMs infected with H37Rv at MOI 5 concentrations over a time-course to (B). The pictures exhibit monolayer identify physiologically relevant integrity and confluency at 48 h. fluctuations. M.tb infections of hMDMs were also conducted with the attenuated The kinetic rates of cAMP secretion strain H37Ra to assess any effects of

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virulence on cAMP production. 72 h after M.tb infection (Fig. 2B). The Supernatants were collected from results demonstrate that M.tb infection triplicate wells for uninfected hMDMs of hMDMs elicits cAMP production in (Fig. 1A) and M.tb-infected a time-dependent fashion. We noted a macrophages at MOI 5 (Fig. 1B). The considerable increase in cAMP at 72 h levels of cAMP remained unaltered 30 post-infection with the attenuated H37Ra min, 1 h, and 3 h upon H37Ra and H37Rv strain compared to the virulent H37Rv infection (Fig. 2A). In contrast, marked strain. increases were observed at 24, 48, and

Figure 2. M.tb induces cAMP at 24, analyses revealed that hMDMs 48, and 72 h post-infection. The transcribe the PDE4A and PDE7A hMDMs were infected with M.tb H37Rv 48 and 72 h post-infection (Fig. or M.tb H37Ra (MOI 5). Supernatants 3A-B). In contrast, gene expression were collected in triplicate at various studies did not detect transcriptional time points, and cAMP levels were alterations for PDE3 and PDE5 (data measured via a competitive binding not shown). The mechanism regarding ELISA. The results are represented as M.tb induction of cAMP remains in mean ± SD. n=1; *P ˂ 0.05, **P ˂ question; however, it is notable that the 0.01, and ***P ˂ 0.001. small decrease in cAMP observed 72 h following infection with the virulent Gene expression of PDE4A and strain H37Rv (Fig. 2B) coincides with PDE7A is increased in hMDMs the increased gene expression of infected with H37Rv. PDE4A and PDE7A at 48 and 72 h. The Inspired by our findings, we proceeded results suggest hMDMs increase the to test the PDE transcription profile of gene expression of PDE4A and 7A as a M.tb-infected hMDMs in comparison to host response to degrade M.tb-induced the increased intracellular cAMP levels. cAMP levels. Quantitative real-time PCR (qRT-PCR)

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Figure 3. Gene expression of PDE4A Gene expression analyses 6, 24, 48, and and PDE7A increases upon M.tb 72 h after infection revealed the infection of hMDMs. The hMDMs expression of specific PDEs. The were infected with H37Rv (MOI 5) in transcription of PDE4A and PDE7A is triplicate. RNA was isolated at the induced 48 and 72 h after infection with indicated time points, and qRT-PCR H37Rv (Fig. 3A-B). These results are analyses were performed to determine possibly linked to the cAMP decrease the gene expression of PDE4A and exhibited by the virulent strain at 72 h PDE7A. Results were normalized to β- post-infection. We speculate that actin and reported as the fold change hMDMs upregulate the gene expression compared to uninfected. n=3; *P < of PDE4A and 7A to degrade cAMP 0.05. levels and prevent H37Rv-mediated dampening of the immune response. 4. Discussion M.tb has numerous interaction points The mechanism(s) underlying the host- along the respiratory tract until AMs pathogen interaction that provokes phagocytose the pathogen. The cAMP activation remains in question. virulence arsenal of M.tb subverts the The enzyme adenylyl cyclase host immune response to establish a canonically regulates cAMP multipronged permissive environment. concentrations. Interestingly, M.tb is Immunosuppressive cyclic nucleotide equipped with 17 adenylyl cyclase second messengers partly influence the genes, and at least one (Rv0386) is immunopathology and intramacrophage involved in the delivery of bacterial growth of M.tb. Preliminary studies cAMP into the macrophage cytoplasm quantifying cAMP in hMDMs revealed [32]. The disruption of cAMP steady- that concentrations increase 24, and 48 state concentrations in macrophages by h following infection with H37Rv, while the Mycobacterial species can interfere H37Ra induces cAMP 48, and 72 h post- with endogenous signal transduction infection (Fig. 2B). The results pathways. Additionally, M.tb is known demonstrate that M.tb infection of to induce host cell Prostaglandin E2 hMDMs elicits cAMP production in a (PGE2) synthesis in the early stages of time-dependent interval. The observed infection. The pleiotropic effects of heterogeneity in cAMP concentrations PGE2 are mediated primarily via the G is potentially attributable to the protein-coupled receptor subtype EP4 in pathogenicity differences of the strains. human macrophages [33]. The action of

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EP4 traditionally stimulates the G primary role of PGE2 is counterintuitive protein α subunit (Gαs), which increases and remains controversial in the intracellular cAMP concentrations by immune regulation of M.tb-infected activating the enzyme adenylyl cyclase hMDMs. Further investigation is [34]. Therefore, it is plausible that M.tb required to elucidate the contribution of infection of hMDMs increases cAMP PGE2, as well as cAMP to the via bacterial secretion and PGE2- intramacrophage growth of M.tb. dependent mechanisms (Fig. 4). The

cyclase by the Gαs promotes the Figure 4. Proposed model. hMDMs catalytic conversion of ATP into cAMP. phagocytose M.tb through complement and pattern recognition receptors. Additionally, the Mycobacterial adenylyl cyclase Rv0386 is associated The host-pathogen interaction provokes with cAMP delivery into the cytosol of an intramacrophage increase in cAMP. hMDMs [32]. PDEs regulate signaling Although the signal transduction events through the hydrolysis of cAMP pathway is incompletely understood, we into AMP. Therefore, PDE gene speculate that PGE2 acts as an expression would be induced as a host extracellular ligand binding to the response mechanism to attenuate M.tb GPCR subtype EP4 in hMDMs. pathogenesis. Activation of the enzyme adenylyl

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The hydrolytic activity of PDE4A and The effect of HDTs can synergistically PDE7A would reestablish cellular enhance pivotal components of the homeostasis by degrading the H37Rv- macrophage response to M.tb. induced cAMP. Marked increases in Activators can reduce cAMP levels act as a virulence factor to immunopathogenesis via critical PDEs, intoxicate human macrophages and while non-specific reactions such as dampen the immune response. cGMP hydrolysis can be inhibited. A Regulation of cAMP would presumably broad spectrum of macrophage restore the delicate balance of responses can be selectively regulated cytokines, chemokines, and lipids in to enhance the intracellular macrophages; thus, enhancing the neutralization of M.tb. The immune response to M.tb. Additionally, immunosuppressive role of PDE4 downstream cAMP-activated signaling inhibition can be clinically relevant in complexes such as PKA will be patients with advanced TB disease. deactivated. The role of PKA in M.tb Granulomas are compact structures that infection includes the inhibition of emerge as a pathological hallmark of phagolysosomal maturation in tissue- chronic M.tb infection. The granuloma resident macrophages and the reduction is composed of macrophages, of IFN-γ secretion in T cells [19,20]. monocytes, dendritic cells, neutrophils, Future studies will characterize all PDE epithelioid cells, multinucleated giant isoforms to elucidate the host-pathogen cells, and a solid layer of lymphocytes interaction during M.tb infection of [36]. The role of granulomas was human macrophages. The gene contemplated as solely protective from expression profile of PDEs in response a histological standpoint. However, to avirulent and virulent M.tb strains chronic inflammation can negatively will provide valuable clues to untangle impact the host through pathological the signaling pathway. lesions inflicting collateral lung damage. The pharmacological PDEs are clinically significant due to inhibition of PDE4 augments the their unique tissue distribution and bactericidal activity of isoniazid and functional properties. The success of reduces pulmonary fibrosis [25,26]. In PDE-specific therapies for different the early stages of M.tb infection, PDEs diseases can be extrapolated as a novel would presumably confer a protective mechanism to regulate macrophage role at the cellular level by degrading activation. The design of HDTs can be cAMP. In contrast, the selective dependent on PDE activator molecules inhibition of PDE4 in chronic TB such as MR-L2 to regulate M.tb patients can reduce tissue damage and pathogenesis. MR-L2 is a pulmonary fibrosis. Further noncompetitive activator promoting the investigation is required to demonstrate reversible dimeric stimulation of PDE4 the efficacy of novel PDE-mediated variants through a phenocopy immunoregulatory HDTs. mechanism [35]. The allosteric activation of PDE4 would result in 5. Acknowledgments lower cAMP intracellular This work was supported by the Texas concentrations due to increased Biomedical Research Institute Cowles degradation, which would favor the host Postdoctoral Fellowship (to CMLW), by reducing M.tb pathogenesis. the Postdoctoral Forum Grant (to CMLW), the National Institutes of Health Grant R01 AI136831 (to LSS)

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and Texas Biomedical Research Funds Role for Alveolar Epithelial (to LSS). I would like to thank Dr. Cells. Front Cell Infect Chrissy M. Leopold Wager, Dr. Microbiol. 2019;9:299. Eusondia Arnett, and Dr. Larry Published 2019 Aug 21. Schlesinger for the mentoring received 6. Pai M, Behr MA, Dowdy D, et al. in the developing stages of my career. It Tuberculosis. Nat Rev Dis was an honor to have been surrounded Primers. 2016;2:16076. by knowledgeable, wise, and supportive Published 2016 Oct 27. scientists. CMLW conducted the M.tb infections of hMDMs in the BSL3 7. Hussell T, Bell TJ. Alveolar facility and demonstrated the ELISA macrophages: plasticity in a technique. Additionally, I would like to tissue-specific context. Nat Rev express the utmost gratitude to my Immunol. 2014;14(2):81-93. former professor at St. Mary’s University, Dr. Ahmad Galaleldeen, for 8. Rajaram MV, Ni B, Dodd CE, continuously inspiring and supporting Schlesinger LS. Macrophage me to attain my full academic potential. immunoregulatory pathways in tuberculosis. Semin Immunol. References 2014;26(6):471-485.

1. Joshi JM. Tuberculosis chemotherapy 9. Schorey JS, Schlesinger LS. Innate in the 21 century: Back to the Immune Responses to basics. Lung India. Tuberculosis. Microbiol Spectr. 2011;28(3):193-200. 2016;4(6):10.1128/microbiolspe c.TBTB2-0010-2016. 2. Sotgiu G, Centis R, D'ambrosio L, Migliori GB. Tuberculosis 10. Guirado E, Schlesinger LS, Kaplan treatment and drug G. Macrophages in tuberculosis: regimens. Cold Spring Harb friend or foe. Semin Perspect Med. Immunopathol. 2013;35(5):563- 2015;5(5):a017822. Published 583. 2015 Jan 8. 11. Bryn T, Mahic M, Enserink JM, 3. Ndlovu H, Marakalala MJ. Schwede F, Aandahl EM, Granulomas and Inflammation: Taskén K. The cyclic AMP- Host-Directed Therapies for Epac1-Rap1 pathway is Tuberculosis. Front Immunol. dissociated from regulation of 2016;7:434. Published 2016 Oct effector functions in monocytes 24. but acquires immunoregulatory function in mature 4. Torrelles JB, Schlesinger LS. macrophages. J Immunol. Integrating Lung Physiology, 2006;176(12):7361-7370. Immunology, and Tuberculosis. Trends Microbiol. 12. Rosenbaum DM, Rasmussen SG, 2017;25(8):688‐697. Kobilka BK. The structure and function of G-protein-coupled 5. Ryndak MB, Laal S. Mycobacterium receptors. Nature. tuberculosis Primary Infection 2009;459(7245):356-363. and Dissemination: A Critical

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13. Tuteja N. Signaling through G 19. Kalamidas SA, Kuehnel MP, protein coupled receptors. Plant Peyron, P, et al. cAMP synthesis Signal Behav. 2009;4(10):942- and degradation by phagosomes 947. regulate actin assembly and 14. Serezani CH, Ballinger MN, fusion events: consequences for Aronoff DM, Peters-Golden M. mycobacteria. J Cell Sci. Cyclic AMP: master regulator of 2006;119(Pt 17):3686-3694. innate immune cell function. Am J Respir Cell Mol Biol. 20. Chung YT, Pasquinelli V, Jurado 2008;39(2):127-132. JO, et al. Elevated Cyclic AMP Inhibits Mycobacterium 15. Degraaf AJ, Zasłona Z, Bourdonnay tuberculosis-Stimulated T-cell E, Peters-Golden M. IFN-γ Secretion Through Type I Prostaglandin E2 reduces Toll- Protein Kinase A. J Infect Dis. like receptor 4 expression in 2018;217(11):1821-1831. alveolar macrophages by inhibition of translation. Am J 21. Boswell-Smith V, Spina D, Page Respir Cell Mol Biol. CP. 2014;51(2):242-250. inhibitors. Br J Pharmacol. 2006;147 Suppl 1(Suppl 16. Aronoff DM, Carstens JK, Chen 1):S252-S257. GH, Toews GB, Peters-Golden M. Short communication: 22. Iffland A, Kohls D, Low S, et al. differences between Structural determinants for macrophages and dendritic cells inhibitor specificity and in the cyclic AMP-dependent selectivity in PDE2A using the regulation of wheat germ in vitro translation lipopolysaccharide-induced system. Biochemistry. cytokine and chemokine 2005;44(23):8312-8325. synthesis. J Interferon Cytokine Res. 2006;26(11):827-833. 23. Chen JJ, Sun YL, Tiwari AK, et al. PDE5 inhibitors, sildenafil and 17. Aronoff DM, Canetti C, Serezani vardenafil, reverse multidrug CH, Luo M, Peters-Golden M. resistance by inhibiting the Cutting edge: macrophage efflux function of multidrug inhibition by cyclic AMP resistance protein 7 (ATP- (cAMP): differential roles of binding Cassette C10) protein kinase A and exchange transporter. Cancer Sci. protein directly activated by 2012;103(8):1531-1537. cAMP-1. J Immunol. 2005;174(2):595-599. 24. Movsesian M, Wever-Pinzon O, Vandeput F. PDE3 inhibition in 18. Peters-Golden M. Putting on the dilated cardiomyopathy. Curr brakes: cyclic AMP as a Opin Pharmacol. multipronged controller of 2011;11(6):707-713. macrophage function. Sci Signal. 2009;2(75):pe37. 25. Ahidjo BA, Bishai WR. Published 2009 Jun 16. Phosphodiesterase inhibitors as adjunctive therapies for

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tuberculosis. EBioMedicine. receptor function and 2016;4:7-8. Published 2016 Feb phagocytosis of this bacterium. J 9. Immunol. 1991;147(6):1983‐ 1994. 26. Subbian S, Koo MS, Tsenova L, et 31. Arnett E, Weaver AM, Woodyard al. Pharmacologic Inhibition of KC, et al. PPARγ is critical for Host Phosphodiesterase-4 Mycobacterium tuberculosis Improves Isoniazid-Mediated induction of Mcl-1 and Clearance of Mycobacterium limitation of human macrophage tuberculosis. Front Immunol. apoptosis. PLoS Pathog. 2016;7:238. Published 2016 Jun 2018;14(6):e1007100. Published 17. 2018 Jun 21.

27. Schlesinger LS, Horwitz MA. 32. Agarwal N, Lamichhane G, Gupta Phagocytosis of leprosy bacilli R, Nolan S, Bishai WR. Cyclic is mediated by complement AMP intoxication of receptors CR1 and CR3 on macrophages by a human monocytes and Mycobacterium tuberculosis complement component C3 in adenylate cyclase. Nature. serum. J Clin Invest. 2009;460(7251):98-102. 1990;85(4):1304‐1314. 33. Takayama K, García-Cardena G, 28. Schlesinger LS. Macrophage Sukhova GK, Comander J, phagocytosis of virulent but not Gimbrone MA Jr, Libby P. attenuated strains of Prostaglandin E2 suppresses Mycobacterium tuberculosis is chemokine production in human mediated by mannose receptors macrophages through the EP4 in addition to complement receptor. J Biol Chem. receptors. J Immunol. 2002;277(46):44147-44154. 1993;150(7):2920‐2930. 34. Sugimoto Y, Narumiya S. 29. Schlesinger LS, Bellinger-Kawahara Prostaglandin CG, Payne NR, Horwitz MA. E receptors. J Biol Chem. Phagocytosis of Mycobacterium 2007;282(16):11613-11617. tuberculosis is mediated by human monocyte complement 35. Omar F, Findlay JE, Carfray G, et receptors and complement al. Small-molecule allosteric component C3. J Immunol. activators of PDE4 long form 1990;144(7):2771‐2780. cyclic AMP phosphodiesterases. Proc Natl 30. Schlesinger LS, Horwitz MA. Acad Sci U S A. Phagocytosis of Mycobacterium 2019;116(27):13320-13329. leprae by human monocyte- derived macrophages is 36. Ndlovu H, Marakalala MJ. mediated by complement Granulomas and Inflammation: receptors CR1 (CD35), CR3 Host-Directed Therapies for (CD11b/CD18), and CR4 Tuberculosis. Front Immunol. (CD11c/CD18) and IFN-gamma 2016;7:434. Published 2016 Oct activation inhibits complement 24.

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Cardiovasc Pharmacol. 37. van der Pouw Kraan TC, Boeije LC, 2012;59(2):116-123. Smeenk RJ, Wijdenes J, Aarden 40. van Driel MF. LA. Prostaglandin-E2 is a potent Fosfodiësteraseremmers: inhibitor of human interleukin effectiviteit en nieuwe 12 production. J Exp Med. toepassingen [Phosphodiesterase 1995;181(2):775-779. inhibitors: effectiveness and new applications]. Ned Tijdschr 38. Hilkens C, Snijders A, Vermeulen Geneeskd. 2006;150(29):1613- H, van der Meide P, Wierenga 1616. E, Kapsenberg M. Accessory cell-derived interleukin-12 and 41. Rajaram MV, Ni B, Dodd CE, prostaglandin E2 determine the Schlesinger LS. Macrophage level of interferon-gamma immunoregulatory pathways in produced by activated human tuberculosis. Semin Immunol. CD4+ T cells. Ann N Y Acad 2014;26(6):471-485. Sci. 1996;795:349-350. 42. Etna MP, Giacomini E, Severa M, 39. Tang EH, Libby P, Vanhoutte PM, Coccia EM. Pro- and anti- Xu A. Anti-inflammation inflammatory cytokines in therapy by activation of tuberculosis: a two-edged sword prostaglandin EP4 receptor in in TB pathogenesis. Semin cardiovascular and other Immunol. 2014;26(6):543-551. inflammatory diseases. J

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