The bronchodilatory drug tiotropium bromide produces respiratory benefits in COPD patients, but apart from reduced cholinergic signaling and altered cholinergic contractile tone, inflammatory pathways affected by the drug remain unknown in detail. Recently published data indicate that tiotropium may decrease airway inflammation and airway remodeling (Santus et al. 2012; Pera et al. 2011). We have previously shown that in patients treated with formoterol + tiotropium there are increased acetylated H3 and H4 histone levels (Holownia et al. 2010, 2013b). Histones are important in inflammatory signaling, because they are responsible for gene transcription and expression of inflammatory and anti-inflammatory proteins. Due to a significant role of local inflammation in COPD, it seems that induced sputum analysis could provide relevant information regarding the intensity of inflammation and the immune mechanisms that are involved. Our data show that in combined therapy there is an inappreciable change in the number of monocytes, but the number of CD8⁺ cells decreases, resulting in a decrease in the CD4⁺/CD8⁺ ratio. It has been shown that a T lymphocyte imbalance is related to the inflammatory response of smokers with established COPD (Tzanakis et al. 2004). It has also been shown that increased CD8⁺ T cells are associated with COPD exacerbations and may contribute to COPD progression. Similar data have been observed in smoking asthmatics (Ravensberg et al. 2013). Since CD8⁺ subpopulations of T lymphocytes appear to play a significant role in COPD, a substantial decrease in CD8⁺ cells in the add-on tiotropium therapy should be considered beneficial. It remains to be established how long this decrease persists after the therapy end.

Concerning the activation phenotypes of sputum cells, we quantified HLA-DR antigen expression and subpopulation of Tregs. We show that HLA-DR is expressed in about 10 % of sputum lymphocytes and significantly higher levels are present in monocytes. In patients treated with F + T, HLA-DR expression on lymphocytes, but not on monocytes, was significantly lower. DR is a marker for immune stimulation and a lower antigen expression may indicate that immune functions related to the antigen presentation may be affected. On the other hand, this change may also reflect an adaptative alteration related to decreased inflammation, when cell activation is no longer necessary. In COPD, airflow limitation has been found to correlate not only with an increased number of CD3⁺ T lymphocytes and CD8⁺ cells but also with increased expression of HLA-DR (O’Shaughnessy et al. 1997). Consequently, it seems that decreased HLA-DR may be related to a decrease in local inflammation.