The Impact of Exogenous Factors on Respiratory Pathogen-Induced Innate Alveolar Macrophage Responses in COPD.

Alveolar macrophages in chronic obstructive pulmonary disease (COPD) have fundamentally impaired innate immune responses to toll-like receptor (TLR) ligands of nontypeable Haemophilus influenzae (NTHI). However, whether dysfunctional inflammatory responses in COPD extend to macrophage interactions with intact respiratory pathogens beyond NTHI has not been explored. Furthermore, the influences of exogenous factors, including active smoking and medications, on pathogen-induced innate immune responses have only begun to be investigated. We hypothesized that distinct alveolar macrophage impairments in COPD are not limited to NTHI TLR ligands and that active smoking and select COPD medications modulate innate responses. Alveolar macrophages, obtained from COPD ex-smokers (n = 32) and active smokers (n = 64) by bronchoalveolar lavage (BAL), were incubated with NTHI, Moraxella catarrhalis, and Streptococcus pneumoniae, and with TLR2 and TLR4 ligands. Elicited IL-8 and TNF-α were measured by multianalyte microsphere flow cytometry to determine proinflammatory responsiveness. Induced IL-8, but not TNF-α, was greater from alveolar macrophages of active smokers compared with ex-smokers, in response to NTHI (p = 0.04), M. catarrhalis (p = 0.003), and S. pneumoniae (p = 0.03). Both IL-8 and TNF-α induction by TLR2 and TLR4 ligands were greater in active smokers. While intergroup NTHI- and M. catarrhalis-induced TNF-α levels were no different, they were notably lower among ex-smokers taking anticholinergic medications (p < 0.04 for each), but not with any other bronchoactive medications. Our results support a paradigm of distinct immunologic responses of COPD alveolar macrophages of ex- and active smokers to diverse respiratory pathogens and highlight a subset of ex-smokers whose diminished alveolar macrophage responsiveness may be associated with anticholinergic agents.

Impaired innate immune alveolar macrophage response and the predilection for COPD exacerbations.

BACKGROUND: Alveolar macrophages (AM) in COPD have fundamentally impaired responsiveness to Toll-like receptor 2 (TLR2) and TLR4 ligands of non-typeable Haemophilus influenzae (NTHI). However, the contribution of innate immune dysfunction to exacerbations of COPD is unexplored. We hypothesised that impaired innate AM responses in COPD extend beyond NTHI to other pathogens and are linked with COPD exacerbations and severity. METHODS: AMs, obtained by bronchoalveolar lavage from 88 volunteers with stable-to-moderate COPD, were incubated with respiratory pathogens (NTHI, Moraxella catarrhalis (MC), Streptococcus pneumoniae (SP) and TLR ligands lipopolysaccharide, Pam3Cys) and elicited IL-8 and TNF-α were measured by microsphere flow cytometry. NF-κB nuclear translocation was measured by colorimetric assay. AM TLR2 and TLR4 expression was determined by immunolabeling and quantitation of mean fluorescent indices. Participants were monitored prospectively for occurrence of COPD exacerbations for 1 year following bronchoscopy. Non-parametric analyses were used to compare exacerbation-prone and non-exacerbation-prone individuals. RESULTS: 29 subjects had at least one exacerbation in the follow-up period (exacerbation-prone) and 59 remained exacerbation-free (non-exacerbation-prone). AMs of exacerbation-prone COPD donors were more refractory to cytokine induction by NTHI (p=0.02), MC (p=0.045) and SP (p=0.046), and to TLR2 (p=0.07) and TLR4 (p=0.028) ligands, and had diminished NF-κB nuclear activation, compared with non-exacerbation-prone counterparts. AMs of exacerbation-prone subjects were more refractory to TLR2 upregulation by MC and SP (p=0.04 each). CONCLUSIONS: Our results support a paradigm of impaired innate responses of COPD AMs to respiratory pathogens, mediated by impaired TLR responses, underlying a propensity for exacerbations in COPD.

Phagocytic dysfunction of human alveolar macrophages and severity of chronic obstructive pulmonary disease.

BACKGROUND: Alveolar macrophages in chronic obstructive pulmonary disease (COPD) have fundamental impairment of phagocytosis for nontypeable Haemophilus influenzae (NTHI). However, relative selectivity of dysfunctional phagocytosis among diverse respiratory pathogens: NTHI, Moraxella catarrhalis (MC), Streptococcus pneumoniae (SP), and nonbacterial particles, as well as the contribution of impaired phagocytosis to severity of COPD, has not been explored. METHODS: Alveolar macrophages, obtained from nonsmokers (n = 20), COPD ex-smokers (n = 32), and COPD active smokers (n = 64), were incubated with labeled NTHI, MC, SP, and fluorescent microspheres. Phagocytosis was measured as intracellular percentages of each. RESULTS: Alveolar macrophages of COPD ex-smokers and active smokers had impaired complement-independent phagocytosis of NTHI (P = .003) and MC (P = .0007) but not SP or microspheres. Nonetheless, complement-mediated phagocytosis was enhanced within each group only for SP. Defective phagocytosis was significantly greater for NTHI than for MC among COPD active smokers (P < .0001) and ex-smokers (P = .028). Moreover, severity of COPD (FEV1%predicted) correlated with impaired AM phagocytosis for NTHI (P = .0016) and MC (P = .01). CONCLUSIONS: These studies delineate pathogen- and host-specific differences in defective alveolar macrophages phagocytosis of respiratory bacteria in COPD, further elucidating the immunologic basis for bacterial persistence in COPD and provide the first demonstration of association of impaired phagocytosis to severity of disease.

Ganglioside-binding specificities of E. coli enterotoxin LT-IIc: Importance of long-chain fatty acyl ceramide.

Bacterial heat-labile (LT) enterotoxins signal through tightly regulated interactions with host cell gangliosides. LT-IIa and LT-IIb of Escherichia coli bind preferentially to gangliosides with a NeuAcα2-3Galß1-3GalNAc terminus, with key distinctions in specificity. LT-IIc, a newly discovered E. coli LT, is comprised of an A polypeptide with high homology, and a B polypeptide with moderate homology, to LT-IIa and LT-IIb. LT-IIc is less cytotoxic than LT-IIa and LT-IIb. We theorized that LT-IIc-host cell interaction is regulated by specific structural attributes of immune cell ganglioside receptors and designed experiments to test this hypothesis. Overlay immunoblotting to a diverse array of neural and macrophage gangliosides indicated that LT-IIc bound to a restrictive range of gangliosides, each possessing a NeuAcα2-3Galß1-3GalNAc with a requisite terminal sialic acid. LT-IIc did not bind to GM1a with short-chain fatty acyl ceramides. Affinity overlay immunoblots, constructed to a diverse array of known ganglioside structures of murine peritoneal macrophages, established that LT-IIc bound to GM1a comprised of long-chain fatty acyl ceramides. Findings were confirmed with LT-IIc also binding to GM1a of RAW264.7 cells, comprised of a long-chain fatty acyl ceramide. Thus, LT-IIc-ganglioside binding differs distinctly from that of LT-IIa and LT-IIb. LT-IIc binding is not just dependent on carbohydrate composition, but also upon the orientation of the oligosaccharide portion of GM1a by the ceramide moiety. These studies are the first demonstration of LT-ganglioside dependence upon ceramide composition and underscore the contribution of long-chain fatty acyl ceramides to host cell interactions.

Impaired Innate COPD Alveolar Macrophage Responses and Toll-Like Receptor-9 Polymorphisms.

BACKGROUND: Dysfunctional innate responses of alveolar macrophages to nontypeable Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae contribute to morbidity in chronic obstructive pulmonary disease (COPD). Our earlier studies discovered impaired COPD alveolar macrophage responses to Toll-like receptor (TLR) ligands of nontypeable H. influenzae and provide rationale for further evaluation of TLR signaling. While the role of TLR single nucleotide polymorphisms is increasingly recognized in inflammatory diseases, TLR single nucleotide polymorphisms in COPD have only recently been explored. We hypothesized that specific TLR polymorphisms are associated with dysfunctional innate immune COPD alveolar macrophage responses and investigated polymorphisms of TLR2(Arg753Gln), TLR4(Thr399Ile; Asp299Gly), and TLR9(T1486C; T1237C). METHODS: DNA was purified from cells of 1) healthy nonsmokers (n = 20); 2) COPD ex-smokers (n = 83); 3) COPD active smokers (n = 93). DNA amplifications (polymerase chain reaction) were performed for each SNP. Alveolar macrophages from each group were incubated with nontypeable H. influenzae, M. catarrhalis and S. pneumoniae. Cytokine induction of macrophage supernatants was measured and the association with TLR single nucleotide polymorphism expression was determined. RESULTS: No significant inter-group differences in frequency of any TLR SNP existed. However both TLR9 single nucleotide polymorphisms were expressed in high frequency. Among COPD ex-smokers, diminished IL-8 responsiveness to nontypeable H. influenzae, M. catarrhalis and S. pneumoniae was strongly associated with carriage of TLR9(T1237C) (p = 0.02; p = 0.008; p = 0.02), but not TLR9(T1486C). Carriage of TLR9(T1237C), but not TLR9(T1486C), correlated with diminished FEV1%predicted (p = 0.037). CONCLUSION: Our results demonstrate a notable association of TLR9(T1237C) expression with dysfunctional innate alveolar macrophage responses to respiratory pathogens and with severity of COPD.