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Streptococcus pneumoniae is a pathogen that causes serious respiratory disease and meningitis in the immunocompromised drug abuse population. However, the precise mechanisms by which drug abuse compromises the host immune defense to pulmonary S. pneumoniae infection is not fully understood. Using a well-established murine model of opiate abuse and S. pneumoniae lung infection, we explored the influence of morphine treatment on the interleukin-23 (IL-23)/IL-17 axis and related innate immunity. Impairment of early IL-23/IL-17 production caused by morphine treatment was associated with delayed neutrophil migration and decreased pneumococcal clearance. Furthermore, morphine treatment impaired MyD88-dependent IL-23 production in alveolar macrophages and dendritic cells in response to in vitro S. pneumoniae cell infection. Moreover, morphine treatment significantly inhibited the S. pneumoniae-induced phosphorylation of interferon response factor 3 (IRF3), ATF2, and NF-κBp65. T-cell receptor δ (TCRδ)-deficient mice showed a decrease in IL-17 production and a severely weakened capacity to clear lung S. pneumoniae infection. Finally, morphine treatment resulted in diminished secretion of antimicrobial proteins S100A9 and S100A8/A9 during early stages of S. pneumoniae infection. In conclusion, morphine treatment causes a dysfunction in IL-23-producing dendritic cells and macrophages and IL-17-producing γδT lymphocytes in response to S. pneumoniae lung infection. This leads to diminished release of antimicrobial S100A8/A9 proteins, compromised neutrophil recruitment, and more-severe infection.
Immunocompromised individuals are at high risk for Streptococcus pneumoniae pulmonary infection (30). Previous studies have shown that opiate abuse causes immunosuppression by disrupting both innate and adaptive components of the immune system (26, 33). Opiate abuse is a critical risk factor for increasing susceptibility and severity of bacterial infection, including S. pneumoniae (31, 32). However, additional work is needed to understand the precise mechanisms by which opiate abuse increases the susceptibility to S. pneumoniae lung infection.
Interleukin-23 (IL-23) has been recently identified as a cytokine closely related to IL-12 (5). The balance between IL-23 and IL-12 controls the outcome of inflammatory responses (16). IL-23 is secreted by activated macrophages and dendritic cells (DCs) and induces memory T-cell proliferation and is the critical factor required for T-cell IL-17 expression in response to bacterial challenge (3). IL-23 release leads to the production of IL-17. Furthermore, IL-17 promotes neutrophilic inflammation by upregulating CXC chemokines and hematopoietic growth factors (13). Several recent studies report the important role of IL-23 and IL-17 in the induction of neutrophil-mediated protective immune response against extracellular bacterial or fungal pathogens, such as Klebsiella pneumoniae (14), Pseudomonas aeruginosa (12), Porphyromonas gingivalis (34), Citrobacter rodentium (20), Bacteroides fragilis (9), and Escherichia coli (27).
Generally, the IL-23/IL-17 axis plays an important role in the host defense against bacterial infections (11). Previous studies have demonstrated that IL-17 is critical for the recruitment of phagocytes that leads to the clearance of S. pneumoniae colonization from the mucosal surface of the nasopharynx (18, 35). However, whether the IL-23/IL-17 axis contributes to modulating the innate immunity in response to S. pneumoniae lung infection has not been addressed. Using a well-established opiate abuse and S. pneumoniae lung infection mouse model (31, 32), we demonstrate that S. pneumoniae induces IL-23 and IL-17 expression in the lungs as early as 2 h following infection. Morphine treatment causes a decrease in both IL-23 and IL-17 synthesis during the early stages of infection, leading to delayed neutrophil recruitment. This results in an increased bacterial burden within the lungs and the initiation of systemic disease.
Pathogen-free B6129PF1 and B6.129P2 T-cell receptor δ (TCRδ−/−) mice were obtained from the Jackson Laboratory (Bar Harbor, ME). Mice were housed in a specific-pathogen-free (SPF) facility under barrier conditions. All animal experiments were done in accordance with the Institutional Animal Care and Use Committee's guidelines at the University of Minnesota.
A murine opiate abuse and pneumococcal pneumonia model has been used extensively by our laboratory as previously described (31, 32). In brief, the virulence of this organism was maintained by subculturing bacteria obtained from the spleens of bacteremic mice and storing them at −80°C until use. For culture growth of the bacteria, S. pneumoniae serotype 3 (ATCC 6303; Rockville, MD) were streaked onto a blood agar plate (Becton, Dickinson and Co.) and grown overnight at 37°C. Typical colonies were picked and inoculated into brain heart infusion (BHI) broth. The culture was incubated for 6 h at 37°C to produce log-phase organisms. The bacteria were pelleted by centrifugation and washed twice in endotoxin-free phosphate-buffered saline (PBS; Invitrogen). The concentration of bacteria was determined spectrophotometrically (OD590) and confirmed by plating serially diluted bacteria onto blood agar plates. Mice were lightly anesthetized with isoflurane (Halocarbon Laboratories, River Edge, NJ) and inoculated with approximately 107 CFU of S. pneumoniae in 50 μl of PBS applied to the tip of the nose and involuntarily inhaled. The animals were held in a vertical position for 1 to 2 min, ensuring migration of the inoculum to the alveoli.
Mice were subcutaneously implanted with either a 75-mg morphine slow-release pellet (National Institute on Drug Abuse [NIDA], Rockville, MD) or placebo pellet (controls) 24 h before S. pneumoniae inoculation. The plasma levels achieved following morphine pellet implantation is in the 200- to 350-ng/ml range at steady state for 7 days. Plasma morphine concentrations in patients that are administered morphine for mild to severe pain are in the 20- to 2,000-ng/ml range, while morphine levels in heroin drug abusers are in the 200- to 2,000-ng/ml range. The doses of morphine used in our studies are well within the range observed in the plasma of patients that are administered morphine for moderate to severe pain and in heroin abusers (8, 10, 21, 25, 28).
To assess the effect of IL-17 administration on bacterial load, 15 ng of recombinant murine IL-17 (R&D Systems) was delivered via the intranasal route, in a volume of 50 μl of sterile PBS, following light isoflurane anesthesia 2 h after infection. Mice were sacrificed 24 h postinfection to collect the lung tissue and bronchoalveolar lavage (BAL) fluid samples as well as blood samples in order to test bacterial burden and dissemination.
The trachea was exposed through a midline incision and cannulated with a sterile 22-gauge Abbocath-T catheter (Abbott). BAL fluid samples were obtained by instilling and collecting 1-ml volumes of cold PBS through the incised trachea. A total of 0.9 ml of lavage fluid was retrieved per mouse. Total cell numbers in the BAL fluid samples were counted from each sample with a hemocytometer (Hausser Scientific, Horsham, PA). The BAL fluid neutrophil count was determined on cytospin preparations stained with a Diff-Quick staining kit (IMEB). The neutrophil population in the lung tissue samples was evaluated by the quantification of myeloperoxidase (MPO) activity (31, 32).
Mice were sacrificed by CO2 asphyxiation at 4, 24, and 48 h postinfection. Blood samples were collected through the heart, and 10 μl of blood was plated onto a blood agar plate and incubated at 37°C overnight to determine the presence of bacteria. BAL fluids were obtained as described above. Lungs were aseptically removed, cleared of blood with cold saline, and homogenized in 2 ml of cold PBS. Serial dilutions of the lung homogenates and BAL fluid samples were plated onto blood agar plates. Plates were incubated at 37°C overnight, and S. pneumoniae colonies were counted.
The following cells were used for the in vitro experiments.
Resident alveolar macrophages (AMs) from mice were obtained via ex vivo lung lavage and resuspended in RPMI 1640 to a final concentration of 1 × 106 cells/ml. Cells were allowed to adhere to plates for 1 h (37°C, 5% CO2), followed by two washes with warm RPMI 1640, resulting in >99% of adherent cells identified as AMs by use of a Diff-Quik stain.
Bone marrow-derived DCs (BMDCs) were generated by following a previously described method (19).
AMs and BMDCs (1 × 106) were treated for 24 h in media containing vehicle (PBS control) or various concentrations of morphine (10 nM to 1 μM; NIDA, Rockville, MD). Then, cells were infected with S. pneumoniae (107 CFU; multiplicity of infection [MOI], 10:1), for various periods of time, depending on the experiment.
To determine whether naltrexone (Sigma) antagonizes morphine's actions, naltrexone was used at a concentration of 10 μM to pretreat cells for 1 h before morphine treatment.
To inhibit the MyD88-dependent signaling, cells were pretreated with MyD88 homodimerization inhibitory peptide or control peptide (Imgenex) at 24 h before in vitro cell infection with S. pneumoniae. The inhibitory peptide contains a sequence from the MyD88 TIR homodimerization domain, which specifically inhibits the MyD88-dependent pathway (17).
The levels of IL-23 and IL-17 in the BAL fluids and lung tissues of S. pneumoniae-infected mice and cell culture supernatant of in vitro infected cells were quantified using cytokine-specific enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions.
Quantification of S100A9 and S100A8/A9 was done using ELISA as previously described by Vandal et al. (29).
For SDS-PAGE and subsequent Western blotting, 1 × 106 adherent BMDCs were lysed using M-PER mammalian protein extraction reagent (Pierce) supplemented with a set of protease inhibitors (Roche) and phosphatase inhibitor cocktails (Sigma-Aldrich). Nuclear and cytoplasmic extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Pierce) according to the manufacturer's instruction. Proteins were separated on an SDS gel and transferred onto a nitrocellulose membrane (Amersham Biosciences). Membranes were washed in Tris-buffered saline-Tween 20 (TBST), blocked for 1 h in StartingBlock blocking buffer, and then incubated overnight at 4°C in the primary antibodies, p-NF-κBp65, p-activating transcription factor 2 (ATF2) (Cell Signaling), and interferon response factor 3 (IRF3; Santa Cruz). The blots were then incubated for 1 h at room temperature with anti-rabbit secondary antibody conjugated with horseradish peroxidase (GE Healthcare). Western blot analysis was conducted according to standard procedures, using SuperSignal chemiluminescence detection substrate (Pierce). Signals were detected by an UltraQuant image acquisition and analysis system. To verify the quality of loading, the blots were reprobed with anti-β-actin (Santa Cruz) or anti-TATA binding protein (Abcam).
Native PAGE for analysis of the IRF3 dimerization experiment was performed as described by others (15). Briefly, 50 μg of nondenatured whole-cell protein extracts were loaded onto a preelectrophoresed 4.5% polyacrylamide gel and separated at 350 V at 4°C. Transfer and Western blotting were done as described above.
Total RNA from lung homogenates and BAL fluid cells was isolated using the RNeasy mini kit (Qiagen). Ten nanograms of total RNA was subjected to two-step reverse transcription-PCR (RT-PCR). Real-time SYBR green PCR analysis was performed for IL-23p19 (sense, 5′-CCAGCGGGACATATGAATCT-3′; antisense, 5′-AGGCTCCCCTTTGAAGATGT-3′) and IL-17 (sense, 5′-TCTCTGATGCTGTTGCTGCT-3′; antisense, 5′-CGTGGAACGGTTGAGGTAGT-3′) mRNA and 18S rRNA. Relative quantification was performed using the ABI Prism 7500 sequence detection system (Applied Biosystems). IL-23p19 and IL-17 transcript levels were normalized to 18S rRNA transcript levels from the same preparations of cDNA.
Data were collected from three independent experiments and expressed as the mean ± standard error of the mean (SEM). Where appropriate, mean values were compared using a paired Student's t test or two-way analysis of variance (ANOVA). A P value of <0.05 was considered statistically significant.
To determine the role of IL-23/IL-17 in host defense against respiratory S. pneumoniae infection, IL-23/IL-17 protein and mRNA levels following S. pneumoniae infection in the lung tissue and BAL fluid samples and cells were quantified. As shown in Fig. Fig.1A,1A, IL-23 was rapidly released as early as 2 h following S. pneumoniae infection in both the lung tissues and BAL fluids, was significantly elevated to peak levels at 4 h, and declined by 24 h postinfection. In the lung tissues, IL-17 was detected within 2 h after S. pneumoniae infection and peaked at 4 h postinfection. This was followed by a general decrease, but the levels remained elevated until 24 h postinfection. In the BAL fluids, IL-17 was not detectable during the early stages of infection (2, 4, and 6 h postinfection) and peaked at 24 h postinfection (Fig. (Fig.1B).1B). Furthermore, mRNA levels of IL-23 and IL-17 in the lung tissues was markedly upregulated by S. pneumoniae infection as early as 2 h and remained elevated at 24 h. Similar to IL-17 protein level, IL-17 mRNA level in the BAL cells was observed only 24 h after S. pneumoniae infection (Fig. 1C and D). Our previous studies found that significant leukocyte recruitment to the lung occurred only after 6 h following S. pneumoniae infection (31, 32), suggesting that both IL-23 and IL-17 were produced by lung resident cells and not by recruited cells in the early stages of S. pneumoniae infection. The rapid release of IL-17 and IL-23 following pneumococcal infection may be due to initial release of preformed cytokines. However, we also observed a rapid increase in IL-23 and IL-17 mRNA levels, so the role of transcriptional activation in the sustained synthesis of the cytokines cannot be ruled out.
To address whether morphine disrupts the IL-23/IL-17 axis, leading to compromised neutrophil recruitment and more-severe infection, morphine- and placebo-treated mice were infected with S. pneumoniae, and IL-23/IL-17, neutrophil migration, and bacterial burden were measured in the lungs at various time points. As shown in Fig. 2A to D, morphine treatment caused a decrease in both IL-23 and IL-17 synthesis during the early stages of infection. This decrease in IL-23 and IL-17 production was associated with delayed and decreased neutrophil recruitment into BAL fluid and lung tissues following S. pneumoniae infection, increased bacterial burden within the lungs, and the initiation of systemic disease. These results indicate that a rapid IL-23/IL-17-mediated neutrophil response participates in the initial control of S. pneumoniae infection. These studies also suggest that morphine treatment causes suppression of the IL-23/IL-17 axis, leading to a decrease in neutrophil recruitment and pneumococcal clearance.
To determine the role of IL-17 in host defense against S. pneumoniae, we performed a rescue experiment using recombinant murine IL-17 (rIL-17; 15 ng/mouse) administered intranasally 2 h after infection, the time at which IL-17 becomes detectable in the lung tissues in this infection model. Mice were sacrificed at 24 h postinfection. Morphine-treated mice showed significantly higher burdens of S. pneumoniae in the lung tissues as well as greater dissemination to the blood 24 h postinfection in comparison with placebo-treated control mice. Administration of IL-17 significantly improved lung antibacterial host defense and reduced bacterial dissemination to the blood (Fig. (Fig.3).3). IL-17 treatment significantly reduced the high bacterial burden observed in morphine-treated mice, further suggesting a critical role for IL-17 in host defenses in the opiate abuse and S. pneumoniae lung infection model.
To directly determine which cell populations among lung resident cells produce IL-23 and IL-17 in response to S. pneumoniae infection and, more importantly, to determine whether morphine suppresses this response independent of the presence of other cell types, primary mouse AMs and BMDCs were treated with morphine or vehicle for 24 h and then infected in vitro with S. pneumoniae (MOI, 10:1). As shown in Fig. Fig.4A,4A, S. pneumoniae infection stimulated IL-23 production in both AMs and DCs. However, the levels of IL-23 produced by DCs were markedly higher than those produced by macrophages. Our results suggest that both AMs and DCs contribute to IL-23 production in response to S. pneumoniae infection. However, DCs may be the primary IL-23-producing cell population responding to S. pneumoniae infection. Furthermore, morphine treatment in the higher dose range (1 μM) inhibited this response in BMDCs, while morphine doses in the 10 or 100 nM range had no effect (Fig. (Fig.4A).4A). Micromolar concentrations of morphine are typical drug abuse doses and concentrations achieved in patients that are prescribed morphine for moderate to severe pain. Plasma levels that are in the nanomolar range are generally observed in patients that receive morphine for mild and acute pain (8, 10, 21, 25, 28). The morphine action on S. pneumoniae-induced IL-23 was abolished by naltrexone, a prototypical opioid receptor antagonist, indicating that classical opioid receptors participate in the inhibition of IL-23 production caused by morphine following S. pneumoniae infection (Fig. (Fig.4B4B).
To determine if TLR-MyD88-dependent signaling is essential for IL-23 production following infection, and whether morphine treatment compromises this signaling in IL-23-producing cells, BMDCs were treated with morphine (1 μM) or vehicle (control), plus MyD88 inhibitory peptide or control peptide, in a dose-dependent manner (10 nM to 100 μM) for 24 h and then in vitro infected with S. pneumoniae. After 6 h of cell infection, cell culture supernatant was collected and used to detect IL-23 release by ELISA. As shown in Fig. Fig.4C,4C, treatment of BMDCs with various doses of MyD88 inhibitor resulted in decreased IL-23 production in a dose-dependent manner, indicating that the IL-23 production induced by S. pneumoniae infection is a downstream event that depends on MyD88 activation. Morphine treatment caused a significant decrease in S. pneumoniae-induced IL-23 production in BMDCs when pretreated with MyD88 control peptide. However, no difference in IL-23 production was observed between morphine- and vehicle-treated BMDCs when pretreated with 100 μM MyD88 inhibitory peptide (Fig. (Fig.4C).4C). This finding implies that morphine treatment impairs MyD88-dependent IL-23 production following S. pneumoniae infection.
To determine if unique signaling pathways are activated by S. pneumoniae infection, thereby accounting for the IL-23 expression, downstream signaling elements from MyD88 were evaluated. To investigate if inhibition of S. pneumoniae-induced IRF3, activating transcription factor 2 (ATF2), and NF-κB activity is a critical mechanism by which morphine inhibits IL-23 production following infection, we examined whether morphine treatment had any effect on the phosphorylation status of IRF3, ATF2, and NF-κB in S. pneumoniae-infected BMDCs. The phosphorylation of IRF3 at its C terminus, particularly on Ser-386 and Ser-396, is required for its dimerization and subsequent translocation to the nucleus (2). As shown in Fig. Fig.4C,4C, treatment of BMDCs with morphine reduced IRF3 phosphorylation and led to decreased induction of IRF3 dimerization in response to S. pneumoniae infection. Additionally, ATF2 and NF-κBp65 phosphorylation was induced, respectively, following S. pneumoniae cell infection. Morphine treatment significantly inhibited S. pneumoniae-induced ATF2 and NF-κBp65 phosphorylation (Fig. (Fig.4C).4C). These results suggest a significant role for MyD88-IRF3, ATF2, and NF-κB signaling pathways in mediating morphine's inhibition of IL-23 production.
Το determine the role of IL-17-producing γδT lymphocytes in S. pneumoniae infection and whether morphine treatment modulates IL-17-producing γδT cells, we compared the production of IL-23/IL-17 in the lungs following S. pneumoniae infection in TCRδ−/− (B6.129P2) mice and strain-, age-, and gender-matched wild-type (WT; B6129PF1) mice. Interestingly, S. pneumoniae infection induced IL-23 production in the BAL fluids and lung tissues of both WT and TCRδ−/− mice. Morphine treatment diminished IL-23 production in the early stages of S. pneumoniae infection (Fig. (Fig.5A).5A). However, significant levels of IL-17 production, induced by S. pneumoniae infection, were detected only in the lungs derived from WT mice and not in the lungs of TCRδ−/− mice. Morphine significantly impaired IL-17 production in S. pneumoniae-infected WT mice. These results suggest that γδT cells are an essential IL-17-producing cell population in response to S. pneumoniae infection and that morphine treatment causes a dysfunction in pulmonary IL-17-producing γδT cells, leading to a defect in innate immunity (Fig. (Fig.5B).5B). Consistent with IL-17 production, γδT-cell-deficient mice have a severely weakened capacity to clear S. pneumoniae lung infection. The number of live bacteria in the lungs was significantly higher (P < 0.01) in TCRδ−/− mice than that in WT mice 24 h postinfection. Morphine treatment in the WT mice resulted in a greater bacterial burden in the BAL fluid and lungs than it did in placebo-treated WT mice. Interestingly, no difference in bacterial burden was observed between morphine and placebo treatment of TCRδ−/− mice (Fig. (Fig.5C).5C). These results clearly reveal that the IL-17-producing γδT cells play a critical role in the host resistance to pneumococcal infection and may be involved in morphine's actions.
To determine whether morphine treatment compromises antimicrobial proteins, S100A8/A9 proteins were quantified in the lung tissue and BAL fluid samples derived from mice infected with S. pneumoniae. As shown in Fig. Fig.6,6, morphine treatment markedly decreased S100A9 and S100A8/A9 production in the early stages of S. pneumoniae infection. Secretion of S100 proteins preceded infiltration of neutrophils into the lung and alveolar space (23). These results suggest that morphine treatment may diminish the host release of antimicrobial proteins S100A8/A9, leading to decreased neutrophil recruitment and more-severe infection.
The current study demonstrates, for the first time to our knowledge, that morphine disrupts the IL-23/IL-17 axis, which diminishes the host release of antimicrobial peptides S100A8/A9 and, consequently, decreases neutrophil recruitment in response to S. pneumoniae infection, thus leading to more-severe infection.
Resident AMs and DCs, along with recruited macrophages and neutrophils, play a key role in the clearance of invading S. pneumoniae (22). While AMs are the primary resident cells in the BAL fluid, both DCs and AMs contribute to first-line cellular defense and cytokine secretion at early stages of infection in the lung tissues. Since AMs are key resident immune cells in both lung tissues and BAL fluid, and the main source of early proinflammatory cytokines released in the lungs, we expected AMs to be the major early source of IL-23 production following S. pneumoniae infection. However, our data show that following S. pneumoniae infection, lung tissue levels of IL-23 were significantly greater than that observed in BAL fluids, indicating that cells other than AMs may be the major early source of IL-23 following S. pneumoniae infection. Consistent with this observation, in vitro infection of DCs and AMs with S. pneumoniae reveals that although both DCs and AMs produce IL-23 in response to infection, DCs' production of IL-23 in response to S. pneumoniae infection was significantly greater than AMs'. These data imply that DCs are the primary early source of IL-23 following S. pneumoniae infection.
IL-17 and IL-17F are produced by αβT cells called Th17 cells, as well as by γδT cells (24). IL-17 is a cytokine that induces neutrophil-mediated inflammation, but its role in protective immunity against S. pneumoniae infection remains unclear. Our studies show that IL-17 expression in the S. pneumoniae-infected lung was detected within a few hours after infection and is IL-23 dependent. Interestingly, γδT-cell-deficient mice have a severely impaired capacity to clear lung S. pneumoniae infection. Moreover, γδT-cell-deficient mice showed a significant reduction in cellular infiltration into the airways and expression of IL-17 in the lung following S. pneumoniae infection (6). Our observations suggest that γδT cells may be a predominant source of IL-17 in the lungs following S. pneumoniae infection. Furthermore, no difference in bacterial outgrowth and dissemination was observed between morphine- and placebo-treated groups in TCRδ-deficient mice. Therefore, we conclude that IL-17-producing γδT cells may be a critical cell population participating in morphine treatment-caused increased susceptibility to S. pneumoniae infection. Our study, therefore, expands on the findings by Lu et al. (18) and Zhang et al. (35) that report that during the natural course of pneumococcal colonization, Th17 cells are critically required for the recruitment of phagocytes that leads to the clearance of colonization in both naive and immune hosts. Our findings further demonstrate that IL-17-producing γδT cells are critical for the innate immune response against S. pneumoniae lung infection.
MyD88 significantly contributes to host defense mechanisms that control colonization of the respiratory tract and prevent invasive infection and proliferation of S. pneumoniae in the blood. MyD88 signaling is required for local cytokine production, leukocyte infiltration, and tissue destruction (1). Our results indicate that IL-23 production in response to S. pneumoniae infection requires MyD88 and that morphine treatment impairs MyD88-dependent IL-23 production. Furthermore, potential binding sites for IRF-3, Sma- and Mad-related protein 3 (SMAD-3), ATF2, IRF7, and NF-κB have been identified in the murine IL-23p19 promoter (2). In both macrophages and DCs, IL-23p19 expression is dependent on the binding of c-Rel and RelA NF-κB to the proximal IL-23p19 promoter (7). More recent promoter analyses reveal that besides a site for NF-κB, regulatory elements for IRF3 and ATF2 at the p19 promoter are essential for promoter activity (2). In this study, we show that morphine treatment decreases S. pneumoniae-induced phosphorylation of IRF3, ATF2, and NF-κBp65, suggesting that inhibition of IRF3, ATF2, and NF-κB signaling pathways is a critical mechanism by which morphine inhibits IL-23 production in S. pneumoniae-infected DCs.
There is accumulating evidence that IL-17 regulates the production of antimicrobial proteins in endothelial and epithelial cells (4). Antimicrobial proteins S100A8 and S100A9 are expressed by neutrophils, monocytes, and activated endothelial and epithelial cells. They regulate macrophage and neutrophil transepithelial migration from the lung tissue to the alveolar space as a host response to S. pneumoniae infection (23). The data presented herein show that morphine treatment decreased S100A9 and S100A8/A9 production during the early stages of S. pneumoniae infection. Decreased secretion of S100A8/A9 proteins was associated with reduced infiltration of neutrophils into the lung and alveolar space. Taken together, our study suggests that morphine treatment diminishes the host release of antimicrobial proteins S100A8/A9, which may lead to decreased neutrophil recruitment in response to S. pneumoniae infection.
Findings from this study indicate that IL-23 is produced by DCs and macrophages within a few hours after exposure to S. pneumoniae. This, in turn, triggers rapid IL-17 responses from lung resident γδT cells. IL-17 may promote the production of antimicrobial proteins S100A8/A9. In our previous studies, we have shown that S. pneumoniae infection results in the early release of MIP-2, KC, IL-1, and tumor necrosis factor alpha (TNF-α) (31, 32), resulting in rapid recruitment of neutrophils to the site of infection. Our current findings indicate that activation of the IL-23/IL-17 axis may be important in driving an early immune response against S. pneumoniae. Morphine treatment disrupts the IL-23/IL-17 axis, leading to diminished host release of antimicrobial proteins S100A8/A9. This results in decreased neutrophil recruitment and more-severe infection. The data generated from this study add to a new understanding of mechanisms by which morphine interferes with pulmonary innate immune defenses against S. pneumoniae infection. This study also helps us to identify potential innate immunity-based therapies and prevention strategies against S. pneumoniae infection in the opioid abuse population.
We greatly thank Philippe A. Tessier (Centre de Recherche en Infectiologie, Centre Hospitalier de l'Université Laval, and Faculty of Medicine, Laval University, Quebec, Canada) for providing the S100A8/A9 and S100A9 ELISA kits.
This work was supported by grants R03 DA023353 (J. Wang) and R01 DA12104, K02 DA015349, and P50 DA11806 (S.R.) from The National Institutes of Health and by funds from the Minneapolis Veterans Affairs Medical Center (R.A.B.).
Editor: J. N. Weiser
Published ahead of print on 7 December 2009.