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.