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Novel relationships of markers of monocyte activation and endothelial dysfunction with pulmonary dysfunction in HIV-infected persons
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"A major strength of our current study is the longitudinal follow-up, which is more strongly suggestive of a relationship between immune cell activation and endothelial dysfunction with reduction in pulmonary function than are prior cross-sectional analyses.
In a well characterized cohort of both HIV-infected and HIV-uninfected men, we found cross-sectional associations of several inflammatory biomarkers (IL-6, TNFα, LPS, sCD163, and IL-2sRα) and the endothelial mediator endothelin-1 with worse pulmonary function in the cohort. Additionally, we identified interactions between HIV seropositivity and two mediators (sCD163 and endothelin-1) with measures of obstruction or diffusing impairment. Finally, we demonstrated that among HIV-infected participants with serial PFT, baseline sCD163 and endothelin-1 continued to be associated with lower pulmonary function in longitudinal analysis.......Features relatively unique to persons with HIV that may contribute to pulmonary pathophysiology include persistent immune activation, microbial translocation, and endothelial dysfunction......A major finding of this study is the novel discovery that endothelin-1 is independently associated with worse airflow obstruction and diffusing capacity impairment in persons with HIV, and that it is associated with lower FEV1 percentage-predicted and lower DLCO percentage-predicted over time. Endothelial dysfunction is a complication of acute and chronic HIV [35,36], and markers of endothelial activation (including cellular adhesion molecules, methylarginines, fibrinogen, and D-dimer) have been associated with cardiovascular disease in HIV [34,37,39,40]. Endothelial damage markers have also been associated with COPD in the general population [42-46].
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Novel relationships of markers of monocyte activation and endothelial dysfunction with pulmonary dysfunction in HIV-infected persons
AIDS June 1 2016
Fitzpatrick, Meghan E.; Nouraie, Mehdi; Gingo, Matthew R.; Camp, Danielle; Kessinger, Cathy J.; Sincebaugh, James B.; Clarke, Andrew; Ries, John W.; Kleerup, Eric C.; Kingsley, Lawrence; Morris, Alison
aDepartment of Medicine, University of Pittsburgh, Pittsburgh
bDepartment of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, California
cDepartment of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh
dDepartment of Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania, USA.
Abstract
Objective: Chronic obstructive pulmonary disease is a common comorbidity in HIV, with prevalence and severity of disease incompletely explained by risk factors such as smoking and age. Unique HIV-associated factors, including microbial translocation, monocyte activation, and endothelial dysfunction, have been described in other comorbidities, but have not been investigated in relation to pulmonary abnormalities in HIV. This study assessed the relationship of these pathologic processes to pulmonary function in HIV-infected and uninfected individuals and determined if relationships were unique to HIV.
Design: Longitudinal observational study.
Methods: Total 274 participants completed pulmonary function testing. Markers of inflammation (IL-6, IL-8, and TNFα), microbial translocation (lipopolysaccharide, sCD14), monocyte activation (sCD163, sCD14, and IL-2 receptor), and endothelial dysfunction (endothelin-1) were measured at baseline. Cross-sectional and longitudinal analyses were performed, adjusting for pertinent covariates.
Results: In HIV-infected individuals, higher IL-6 and endothelin-1 associated with worse forced expiratory volume in one second (FEV1) percentage-predicted, and higher sCD163 associated with worse FEV1/forced vital capacity. IL-6, TNFα, lipopolysaccharide, sCD163, IL-2 receptor, and endothelin-1 associated with diffusing impairment. sCD163 and endothelin-1 interacted with HIV status in relationship to pulmonary function. In HIV-infected individuals only, baseline endothelin-1 was associated with lower FEV1, and sCD163 and endothelin-1 were associated with lower diffusing capacity during follow-up.
Conclusion: Circulating markers of HIV-associated humoral abnormalities are associated with airflow obstruction and diffusing impairment and baseline measures of monocyte activation and endothelial dysfunction associate with lower pulmonary function over time in HIV-infected persons. These findings suggest mechanisms of the disproportionate burden of chronic obstructive pulmonary disease in HIV-infected persons.
Introduction
Abnormal pulmonary function is well documented in persons with chronic HIV, and specific pulmonary diseases such as chronic obstructive pulmonary disease (COPD), asthma, and pulmonary vascular disease are also disproportionately frequent [1-15]. HIV appears to be an independent risk factor for these diseases, as these associations have been demonstrated despite consideration of known risk factors for chronic pulmonary disease such as smoking, prior pneumonia history, or injection drug use [2-4,7,14]. The biologic mechanisms that underlie pulmonary function abnormalities in this vulnerable population remain incompletely described.
Features unique to chronic HIV infection are likely to contribute to the unexplained differences in pulmonary disease incidence, prevalence, and progression between HIV-infected persons and the general population. One potential contributor is enhanced inflammation, generally in the context of persistent immune activation [16-18]. We have recently demonstrated cross-sectional relationships between peripheral T-lymphocyte activation and senescence and classic inflammatory markers IL-6 and C-reactive protein with pulmonary function testing (PFT) abnormalities in HIV [19], but longitudinal analyses are lacking. Additionally, these markers explain only a small percentage of the variance in pulmonary function among HIV-infected persons. Related and alternative processes characterizing chronic HIV that would plausibly associate with COPD include monocyte activation, microbial translocation, and endothelial dysfunction. These conditions have been related to other HIV-associated non-AIDS comorbidities, but have not been explored in HIV-associated pulmonary disease.
Abnormally elevated measures of monocyte activation, which persist despite antiretroviral therapy (ART), have been linked to HIV progression, mortality, and HIV-associated cardiovascular and neurocognitive conditions [20-29]. Given the centricity of the alveolar macrophage to pulmonary immunity, monocyte activation may be expected to relate to HIV-associated pulmonary disease. The potential causes of immune activation (T-lymphocyte or monocyte) in HIV are myriad, but one potential-specific driver is microbial translocation of bacteria across the gut epithelial barrier, with bacterial cell wall elements serving as immunogenic triggers [30-33]. Finally, endothelial damage and dysfunction are consequences of HIV infection that have been associated with cardiovascular and other HIV-associated comorbidities [34-40]. As translational and epidemiologic data suggest a link between endothelial damage and apoptosis with COPD in the general population [41-46], endothelial factors may be expected to play a role in PFT abnormalities in persons with HIV.
We investigated these three constructs (microbial translocation, macrophage activation, and endothelial dysfunction) in conjunction with classic inflammatory markers to describe their relationships to one another and to determine their potential role in HIV-related pulmonary disease. We measured markers of Th1 inflammation (IL-6, IL-8, and TNFα), microbial translocation [sCD14, lipopolysaccharide (LPS)], monocyte activation [sCD163, sCD14, and IL-2 soluble receptor α (IL-2sRα)], and endothelial dysfunction [endothelin-1] in a prospective cohort of HIV-infected and HIV-uninfected men. Using cross-sectional and longitudinal analyses, we determined which biomarkers independently associated with PFTs in HIV-infected participants, to what degree biologic markers from related pathways correlate in this population, and whether specific associations were unique to HIV-infected versus HIV-uninfected persons.
Discussion
In a well characterized cohort of both HIV-infected and HIV-uninfected men, we found cross-sectional associations of several inflammatory biomarkers (IL-6, TNFα, LPS, sCD163, and IL-2sRα) and the endothelial mediator endothelin-1 with worse pulmonary function in the cohort. Additionally, we identified interactions between HIV seropositivity and two mediators (sCD163 and endothelin-1) with measures of obstruction or diffusing impairment. Finally, we demonstrated that among HIV-infected participants with serial PFT, baseline sCD163 and endothelin-1 continued to be associated with lower pulmonary function in longitudinal analysis.
Prior studies have demonstrated associations between chronic HIV infection and pulmonary dysfunction; with reports of more frequent respiratory symptoms [14], worse diffusing capacity [4,7], and more frequent COPD diagnoses and radiographic emphysema [1-3,14,54] among persons with HIV. These observations are in part related to known exposures, most notably high frequency and intensity of cigarette smoking [55,56], but analyses in well conducted studies have established that a gap persists between HIV-infected and HIV-uninfected groups independent of age, smoking, and prior pulmonary infection [1-3,14,54]. We have previously shown an association between systemic inflammation and T-lymphocyte activation with HIV-associated COPD [19], but data are lacking regarding integration with specific pathways. Features relatively unique to persons with HIV that may contribute to pulmonary pathophysiology include persistent immune activation, microbial translocation, and endothelial dysfunction.
A major finding of this study is the novel discovery that endothelin-1 is independently associated with worse airflow obstruction and diffusing capacity impairment in persons with HIV, and that it is associated with lower FEV1 percentage-predicted and lower DLCO percentage-predicted over time. Endothelial dysfunction is a complication of acute and chronic HIV [35,36], and markers of endothelial activation (including cellular adhesion molecules, methylarginines, fibrinogen, and D-dimer) have been associated with cardiovascular disease in HIV [34,37,39,40]. Endothelial damage markers have also been associated with COPD in the general population [42-46]. Interestingly, parallel associations between endothelin-1 and PFTs were not observed in the HIV-uninfected participants, despite similar median levels of endothelin-1 in HIV-infected and HIV-uninfected persons. The lack of association in the HIV-uninfected subset is unlikely to be explained by lower statistical power because of sample size or precision relative to HIV-infected, given that the HIV-uninfected group was larger, and the statistical variances of affected PFT values were similar between the two groups. Interaction testing confirmed significant interactions between endothelin-1 levels and HIV serostatus with postbronchodilator FEV1 percentage-predicted and DLCO percentage-predicted in this cohort, supporting a larger effect size of endothelin-1 relative to PFT outcomes in HIV-infected persons.
The finding of circulating endothelin-1 in association with PFT abnormalities adds to the existing literature describing a relationship between endothelial dysfunction and pulmonary disease, though in our cohort the association was only seen in HIV-infected persons. In a small study of individuals with COPD, plasma endothelin-1 negatively correlated with FEV1, and endothelin-1 was increased in sputum and plasma during exacerbations, compared to controls during clinical stability [45]. Additionally, endothelin-1 is increased in exhaled breath condensate in exercise-induced bronchospasm and asthma [57], and has been related to the development of bronchiolitis obliterans syndrome in pulmonary allografts [58]. Recently, a very large population study (not selecting for persons with respiratory disease) found endothelin-1 levels to negatively correlate with FEV1[44]. Other investigations have related pulmonary outcomes to vascular measures of endothelial function; one such study demonstrated cross-sectional associations between brachial artery flow-mediated dilation, FEV1, and quantitative CT emphysema scores [41]. Another recent investigation found a relationship between quantitative CT measure of lung total tissue volume and DLCO, further supporting that the pulmonary microvasculature plays a significant role in the gas exchange abnormalities of COPD [59].
The drivers of endothelin-1 expression are not completely elucidated, but include reactive oxygen species, inflammatory mediators, and hypoxia-related factors [60]. In addition to its most clearly defined function as a potent vasoregulator, endothelin-1 is a bronchoreactive mediator [61,62] and an inflammatory regulator, having been found in vivo to affect regional expression of IL-6 [63,64], monocyte chemoattractant protein-1 [63], CX3CL1 [65], and other inflammatory cytokines [66]. Animal and in-vivo studies suggest potential mechanisms of endothelial-pulmonary associations, including emphysema secondary to endothelial apoptosis and endothelial inflammation, regional inflammation and chemotaxis, and matrix metalloproteinase production [43,46,67-70].
The greater relationship between endothelin-1 and PFTs in HIV-infected persons, despite similar circulating endothelin-1 levels to HIV-uninfected persons in the cohort, is not explained. It is possible that persons with HIV demonstrate more local variation in endothelin-1 regulation or receptor expression - for instance, one study has found that in response to vasoreactive challenge, local endothelin-1 remained elevated in persons with hypertension, but not in healthy volunteers, suggesting regional dysregulation with lack of adaptation [71]. Whether a similar local adaptation failure may be present in HIV is unknown. Additionally, the source of elevated circulating endothelin-1 in our participants is not known. Although pulmonary vascular endothelium is the most common location of endothelin-1 synthesis, endothelin-1 can be produced by many cell types, notably epithelium, smooth muscle, and immune cells, including alveolar macrophages [72,73]. Endothelin-1 expression from alveolar macrophages has been shown to be increased in congestive heart failure patients, although the enhanced elaboration from macrophages did not correlate with serum levels [73]. Future studies measuring endothelin-1 levels from the pulmonary compartment or assessing regional endothelin-B receptor expression may help explain the mechanism of its differential effect in HIV-infected versus HIV-uninfected persons.
The second novel finding of this study, equally important, is the association between markers of monocyte activation with pulmonary function in persons with HIV. Monocyte activation is a feature of both acute and chronic HIV infection [20] and is an important correlate of HIV-associated morbidity in the ART era [21]. Commonly measured markers of monocyte activation include sCD163, a circulating molecule which is enzymatically cleaved from monocytes upon activation [74], and sCD14. Both markers are elevated in HIV infection, including among persons with viral control [24,25,27,28]. Additionally, both are increased in inflammatory HIV-associated non-AIDS conditions such as cardiovascular disease, atherosclerosis [20,29,75-78], and HIV-associated neurocognitive disorders [22,23,26,79]. Soluble IL-2 receptor α (IL-2sRα, or sCD25), the circulating cleavage product of the IL-2 receptor (CD25), is a marker of disease activity in chronic inflammatory diseases [80-82]. CD25 is present on many leukocytes, including activated T-lymphocytes, and IL-2sRα has thus been used as a circulating surrogate of T-lymphocyte activation [83,84]; as such, it has been found to associate with cardiovascular risk among women with HIV [84]. Of interest, however, monocytes also elaborate CD25, and IL-2sRα levels have been associated with percentage of nonclassical monocytes while failing to associate with T-lymphocyte subsets [81]. In this cohort, regardless of HIV serostatus, IL-2sRα significantly correlated with sCD163 and sCD14, suggesting that it may be linked to monocyte activation. Given the importance of macrophages to pulmonary immunity and their relationship to COPD [85], monocyte contributions to pulmonary dysfunction may also be significant among persons with HIV, but these markers had not previously been investigated with reference to PFTs.
In the current study, median sCD163, sCD14, and IL-2sRα values were significantly higher in the HIV-infected than HIV-uninfected group, in keeping with prior data [24,28,29,84]. sCD163 independently associated with worse pulmonary dysfunction, including lower FEV1/FVC postbronchodilator ratio, greater odds of bronchodilator responsiveness, and lower DLCO percentage-predicted in HIV-infected participants. Similar associations were not demonstrated in the HIV-uninfected group, though there were fewer persons with a bronchodilator response (9/150 [6%] vs. 13/124 [11%]), which reduced power to detect an association. Nonetheless, a statistical interaction was detected between HIV seropositivity and positive bronchodilator response. IL-2sRα was also associated with DLCO percentage-predicted among HIV-infected persons in this cohort, but unlike sCD163, relationships were nearly identical in the HIV-uninfected group. Importantly, sCD163 baseline levels also were associated with lower DLCO over the period of follow-up, though no significant longitudinal association was found with FEV1. Exploratory path analyses supported that the effect of sCD163 on FEV1 and DLCO percentage-predicted are at least partially mediated by endothelin-1, suggesting that monocyte activation and endothelial dysfunction may be linked with regard to pulmonary disease in HIV-infected persons. These findings require further exploration in mechanistic studies.
Of the humoral inflammatory markers investigated, IL-6 and TNFα were higher in HIV-infected individuals with worse pulmonary function. Higher plasma IL-6 levels were associated with both lower postbronchodilator FEV1 percentage-predicted and diffusing impairment and, and TNFα was associated with bronchodilator response and lower DLCO percentage-predicted. Relationships were similar between HIV-infected and HIV-uninfected participants. IL-6 and TNFα levels were closely correlated in this cohort, suggesting they represent the same Th1 inflammatory pathway. Consistent with findings in some general population COPD analyses [86,87], these markers associated with spirometry in the HIV-uninfected group. We have found similar associations with IL-6 and with C-reactive protein (not measured in this study) with PFTs in a different HIV-infected cohort [19], and these inflammatory markers have been associated with other HIV-associated chronic diseases, including cardiovascular disease [17,88], and with COPD outcomes in case of IL-6 [87,89]. One prior HIV study comparing IL-6 to radiographic emphysema found no relationship between the two, though that study did not include PFTs to allow for direct comparison to our data [54]. Notably, despite previous findings of IL-8 associating with elevated pulmonary artery systolic pressure and dyspnea in HIV-infected persons [12], IL-8 did not significantly associate with PFTs in this cohort. This finding is in keeping with our previous data in a different HIV-infected cohort [19] and with available COPD literature [86].
Microbial translocation is a feature of HIV infection [33] and has been linked to chronic immune activation despite ART [30-32]; because of this association with systemic inflammation, potential for contribution to pulmonary disease, particularly COPD, is compelling. A previous investigation of potential microbial translocation markers in persons with HIV found an independent association between sCD14 and risk of radiographic emphysema, though this study did not assess spirometry [54]. In our analysis, we measured circulating levels of microbial translocation markers LPS and the soluble form of its ligand (sCD14). LPS was associated with lower DLCO in both HIV-infected and HIV-uninfected groups, but there was no association with obstruction. There were no associations between sCD14 levels and any pulmonary function outcome in either HIV-infected or HIV-uninfected participants. In the current study, with mostly well controlled HIV (81% of HIV-infected participants virally suppressed, 2% with CD4+ T-cell counts <200 cells/μl), LPS and sCD14 did not correlate. This lack of association suggests that in this cohort, sCD14 does not specifically represent the construct of 'microbial translocation,' but may represent nonspecific monocyte activation, as discussed above. Although prior microbial translocation data have demonstrated that high levels of sCD14 in the plasma reflect LPS exposure [30], the two markers have not been reliably correlated in persons without significant immune suppression [90]; furthermore, in-vivo work has shown IL-6 or IL-1β stimulation can induce sCD14 production comparable to that induced by LPS [91], suggesting inflammatory signaling independent of microbial translocation. Microbial translocation cannot be discounted as a factor in HIV-associated pulmonary disease based on this relatively small sample, but the contributions compared to other pathways appear to be limited.
We did not find differences in PFT decline (FEV1 or DLCO percentage-predicted) between HIV-infected and HIV-uninfected participants in this cohort, possibly because of the relatively limited number of participants who returned for serial testing. Our results were similar to those from another longitudinal cohort, where no differences were found in longitudinal spirometry between HIV-infected and HIV-uninfected groups unless participants were analyzed in strata of poor viral control or CD4+ cell deficiency [92]. We did not have an adequate number of poorly controlled participants to independently assess this factor in this study. In addition, because the Multicenter AIDS Cohort study was started in the 1980s, the cohort may suffer from survivor bias, and current individuals may have better pulmonary function than those who have died.
The study has several limitations. First, only circulating biomarkers were assessed. Evaluation of biomarker and receptor concentrations from respiratory samples or human bronchial epithelial cells with similar correlations to circulating markers and to pulmonary function will be revealing regarding systemic and local relationships of these molecules. Additionally, complementary vascular mediators (such as endothelial growth factors, cellular adhesion molecules, and methylarginines) and other markers of endothelial apoptosis, such as endothelial microparticles, were not examined in this cohort. Further studies of the associations between levels of endothelin-1 and other vascular mediators and pulmonary outcomes in HIV-infected populations are warranted. A final important limitation of this study is that the findings are limited to men. COPD phenotypes and pathogenesis demonstrate sex variation [93,94]; additionally, biomarker levels and activity may vary between men and women. Endothelin-1 demonstrates sex differences in the cardiovascular system [95] and kidney [96], and sCD163 has been found to be higher in women [97,98], with possible modulation by sex hormones [99]. These associations require confirmation in a cohort, including female participants.
A major strength of our current study is the longitudinal follow-up, which is more strongly suggestive of a relationship between immune cell activation and endothelial dysfunction with reduction in pulmonary function than are prior cross-sectional analyses. Additionally, including both HIV-infected and HIV-uninfected participants allowed direct comparison between groups and identification of pathophysiology that may be particularly enhanced in persons with HIV. Finally, assessing multiple biomarkers in one cohort permitted comparison of their relationships, allowing hypothesis-generating inferences for possible interactions between biologic pathways. In conclusion, this study discovered two new potential arenas contributing to the disproportionate burden of pulmonary dysfunction among persons with HIV. Further exploration of monocyte activation and endothelial dysfunction in the role of HIV-associated lung disease and symptoms may lead to new implications for treatment in this population.
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