Tropheryma whipplei colonization in HIV-infected individuals is not associated with lung function or inflammation

Abstract

Studies demonstrate that Tropheryma whipplei (T. whipplei) is present in the lungs of healthy individuals without acute respiratory symptoms or acute respiratory infection and is more common in the lungs of HIV-infected individuals and in smokers. The impact of T. whipplei colonization in the lung on local inflammation and pulmonary dysfunction in HIV-infected individuals is currently unknown. In this study, we performed specific polymerase chain reaction (PCR) and sequencing for T. whipplei in bronchoalveolar lavage (BAL) and induced sputum (IS) samples in 76 HIV-infected participants from three clinical sites. Pulmonary function and proinflammatory cytokine and chemokine levels in BAL were measured. Frequency of T. whipplei in either BAL or IS was 43.4%. The sensitivity and specificity of IS compared to BAL for detection of T. whipplei was 92.3% and 84.2%, respectively, and isolates of T. whipplei in the BAL and IS in the same subject shared genetic identity. Pulmonary function measures were not associated with T. whipplei colonization, and proinflammatory cytokine and chemokine levels in BAL and plasma as well as percentages of inflammatory cells in BAL and IS were not higher in colonized individuals. Overall, these results indicate that T. whipplei colonization in the lung is common, but may not be associated with decreased pulmonary function or inflammation in HIV-infected individuals.

Fig 1. Partial hsp65 gene sequence of T. whipplei in bronchoalveolar lavage and induced sputum samples from the same participant.

The gene sequences of the reference T. whipplei hsp65 and the isolates of T. whipplei from number 5 participant’s BAL and IS were analyzed using Molecular Evolutionary Genetics Analysis version 6 (MEGA6) software packages. Both isolates of T. whipplei in the BAL and IS in the same participant had mutations in the hsp65 gene and shared genetic identity. BAL, Bronchoalveolar lavage; IS, induced sputum; hsp65, heat-shock protein 65 gene.

Fig 2. Measures of pulmonary function in HIV-infected individuals by T. whipplei status.

Post-bronchodilator spirometry and diffusing capacity for DLco were performed for each participant per American Thoracic Society guidelines. Measures of pulmonary function were analyzed by T. whipplei status using Kruskal-Wallis test for continuous variables and Fisher exact test for categorical. There were no significant differences in pulmonary function test parameters by T. whipplei status. FEV1, forced expiratory volume in one second; FVC, forced vital capacity; DLco, diffusing capacity for carbon monoxide.

Fig 3. Levels of proinflammatory biomarkers in bronchoalveolar lavage by T. whipplei status.

Levels of proinflammatory cytokines and chemokines in BAL supernatant were measured using a Luminex assay kit from Bio-Rad per the manufacturer’s protocol. Data were analyzed by T. whipplei status using Kruskal-Wallis test for continuous variables and Fisher exact test for categorical. There were no significant differences in levels of cytokine and chemokine by T. whipplei status.IL-1RA, interleukin-1 receptor antagonist.

Fig 4. Levels of proinflammatory biomarkers in plasma by T. whipplei status.

Levels of proinflammatory cytokines and chemokines in plasma were measured using a Luminex assay kit from Bio-Rad per the manufacturer’s protocol. Data were analyzed by T. whipplei status using Kruskal-Wallis test for continuous variables and Fisher exact test for categorical. There were no significant differences in levels of cytokine and chemokine except IL-1RA by T. whipplei status. IL-1RA, interleukin-1 receptor antagonist. *P = 0.025.

Fig 5. Cytology of bronchoalveolar lavage and induced sputum samples by T. whipplei status.

Differential cell counts in BAL fluid and IS were performed to determine the numbers and percentages of inflammatory cells. Data were analyzed by T. whipplei status using Kruskal-Wallis test for continuous variables and Fisher exact test for categorical. There were no significant differences in percentages of different inflammatory cell types by T. whipplei status.