Discussion

To date, few studies have repeatedly examined the local inflammation in the lungs in patients with sarcoidosis and to our knowledge, this is the first study exploring the effect of infliximab on BALF cells.
We found that 10/13 patients with sarcoidosis refractory to conventional treatment benefitted from therapy with either improvement of radiographic extent of sarcoidosis related changes, or stabilization while concomitant immunosuppressant therapy could be decreased. This was paralleled by a statistically significant reduction of CD4/CD8 ratio and expression of the activation marker CD69 on CD4+ T-cells, indicating a decreased CD4+ T-cell alveolitis. Furthermore, the number of mast cells decreased significantly in responders but no significant changes were seen in cell concentration, percentage and absolute numbers of lymphocytes. The percentage of FoxP3+ CD4+ T-cells decreased in all patients except for two.
There is still a controversy on how to define response in pulmonary sarcoidosis. In a consensus document from 201235 it is suggested that disease progression or regression is defined as a ≥15% change in FVC or a 5-15% change in FVC in association with a definite change in chest radiographic extent. However, the largest clinical trial on infliximab therapy reported an average improvement of only 2.5% from baseline in percent of predicted FVC36. Later studies have used different endpoints. One study from 2014 used improvement >10% in either FVC, FEV1 or DLCO but found that only 56.5% of 69 patients with respiratory functional impairment fulfilled that criteria after one year or longer with TNF-α inhibitor treatment (infliximab or adalimumab)37. Another study from 2015 used improvement ≥5% FVC percent of predicted, a 40% reduction in biomarker levels or maximum standardized uptake value on18F –fluorodeoxyglucose positron emission tomography and assessment of quality of life as markers for response in a composite index. The majority of 48 included patients fulfilling 26 weeks infliximab therapy had a pulmonary treatment indication and mean FVC percent of predicted improved 6.64%23. In our study population 4/10 responders do not fulfill the criteria suggested by the 2012 consensus35. However, considering that included patients had a deteriorating disease despite conventional treatment, and that several previous studies on infliximab therapy have found improvements smaller than suggested in the 2012 consensus, and that the infliximab treatment in the four not fulfilling these criteria led to a stabilization of the disease despite reduction in concomitant immunosuppressant treatment, we think it is reasonable to define them as responders. Similarly, the non-responders did not decrease that much in FVC, but disclosed a clear progress on CT, and needed more immunosuppressant, hence we think they should be defined as non-responders.
Sarcoidosis is characterized by an exaggerated T-cell immune response, which is believed, at least to some extent, to be the result of dysfunctional Tregs. However, high numbers of mast cells in the lavage fluid have been connected to a more active and severe sarcoidosis, and mediators released from activated mast cells have been suggested to be involved in development of fibrosis15,17. Interestingly, mast cells can be activated by activated but not resting T-cells, and this has been suggested to be a mechanism underlying the propagation of T-cell mediated inflammatory processes38. Also, Tregs can interact with mast cells, but normally in order to dampen inflammation. However, if the Tregs are defective, mast cell mediated inflammation can be enhanced39. The importance of mast cells is further strengthened by the fact that they are capable of producing TNF-α12,13 and they can also induce CD8+T-cell activation, proliferation and cytotoxicity40. Furthermore, a persistent increase of mast cells on serial BALF measurements has been associated with active disease.
Taken together, with our finding that the mean number of mast cells decreased in responders but increased in both non-responders, it is tempting to speculate that the CD4+ T-cell alveolitis including Treg dysfunction, can lead to an increase in mast cell numbers and activity, further propagating the inflammation by secretion of cytokines, i.e. TNF-α and activation of CD8+ T-cells. Mast cell secreted TNF-α can also be chemotactic for T-cells41and thereby contribute to a propagating T-cell alveolitis in the lungs of patients with non-resolving sarcoidosis.
Only few studies examined how TNF- α exerts its effects in sarcoidosis and whether lung immune cells change during the natural course of the disease. A previous study showed that patients recovered from Löfgren´s syndrome disclosed a termination of the CD4+ T-cell alveolitis, demonstrated by a normalized CD4/CD8 ratio, cell concentration and percentage of lymphocytes42.
When it comes to the effects of TNF-α on immune cells, most studies have focused on Tregs. In cell culture from mice, exposure to exogenous TNF-α promotes Tregexpansion43, but it has also been shown in patients with rheumatoid arthritis that FoxP3 is dephosphorylated, leading to suppression of Treg function44. Treatment with infliximab restored the suppressive capacity of peripheral Tregs and gave rise to a newly differentiated Treg population in RA after 4-6 months45. One study investigated the effect of infliximab on peripheral Tregs in sarcoidosis and in contrast to the situation in RA, the relative frequencies of Treg population decreased in both responders and non-responders after 26 weeks26, which is in line with our results from BALF as we saw a decrease in most patients, also in the two non-responders. The effect on Tregs of infliximab therapy thus remains elusive.
It also needs to be mentioned that the response of infliximab has been suggested not solely to be due to an effect on Tregs. Both changes in soluble TNF receptor 2 expression, functionality of T effector cells and activity of monocytes/ macrophages in blood26,46 have been associated with response to treatment. Also, CD8+ T-cells are influenced by infliximab treatment and this might explain the increased risk for tuberculosis during anti-TNF-α treatment47. Interestingly, CD8+ T-cell activities can also modulate mast cell activities40.
We did not detect any significant changes in cell concentration, percentage and absolute numbers of lymphocytes between base-line and follow-up, as seen in patients having recovered from Löfgren´s syndrome42. Considering the remaining lymphocytosis despite treatment, and the high risk of relapse after cessation of treatment with infliximab, the treatment does not seem to interfere with the primary event causing the inflammation, but rather blocking downstream events in the inflammatory cascade.
Major limitations of this study include a relatively small study sample with a gender imbalance. Especially, results from the non-responders must be interpreted with caution, since they were only two. We can´t be sure that we properly identified the Treg population using FoxP3, as no definitive surface marker that uniquely isolates Treg cells from other T cell populations exists. However, FoxP3 is essential for the function and has been widely used for identification of Tregs48,49.
Major strengths include exploration of the lung inflammation with bronchoscopy before and after treatment, the homogeneity of subjects; all having a non-Löfgren´s syndrome with pulmonary involvement and several years of disease duration.