Discussion
In the present study we report differences in circulatory B cell
subsets, monocyte subsets and IL-6 levels in IgAN patients, compared to
healthy controls (HC) and/or disease controls i.e. patients with ADPKD.
We also found associations between sCD40L levels and the proportion of
pre-switched B cells, as well as sCD40L and MCP-1 levels and albuminuria
in IgAN patients.
B cell development is an important subject to understand the
pathophysiology of IgAN. IgD is expressed on naïve B cells before they
are exposed to antigen and perform class-switching. IgD together with
IgA are the only human immunoglobulins undergoing O-glycosylation (9,
10). Changes in IgA O-glycosylation occur late during B cell maturation
and imply production of Gd-IgA1 (Hit 1) and autoantibodies against
Gd-IgA1 in IgAN (Hit 2) (4, 10), both events crucial in the pathological
process (4, 11, 12). Several immunological effector pathways are
involved and orchestrate these events, for example T-lymphocytes, APCs
and the cytokine/chemokine network. Given this immunological network, we
aimed to simultaneously identify different B cell subsets together with
T-cell, monocyte and cytokine/chemokine signatures with a method that
minimize ex vivo manipulation.
We observed a significant lower proportion of pre-switched B cells and a
higher trend in the naïve B cell population in IgAN compared to both HC
and ADPKD. To validate these observations, we analyzed individual ratios
between naïve and pre-switched B cells and disclosed an increased ratio
in IgAN compared to HC and ADPKD. This may suggest an altered balance in
B cell maturation in IgAN which could reflect a migration of
pre-switched B cells from the circulation to for example follicles in
the gut mucosa and Payer´s patches for further maturation and
class-switching (13).
The proportions of plasmablasts in the peripheral circulation of IgAN
patients were lower compared to HC. Plasma cells constitute an important
B cell subset responsible for antibody production and their life-cycle
in IgAN has been debated. Our data indicate a translocation of
circulatory plasmablasts to lymphoid tissues for further maturation into
antibody-producing cells. This assumption is supported by data that
demonstrate migration of plasma cells to central lymphoid tissue, such
as the bone marrow where they continue to produce underglycosylated
antibodies (14, 15). A lower number of polymeric IgA-secreting plasma
cells in the duodenal mucosa but higher in systemic sites, for example
in the bone marrow have been reported, which support a dynamic view of
the cellular compartmentalize process in IgAN (4, 16). A lower
proportion of circulating plasmablasts in IgAN patients has also
recently been reported by Cols et al (17). And their cohort was
comparable to ours in terms of age and eGFR. However in the study by Si
and co-workers the percentage of plasmablasts was elevated in IgAN (18).
In contrast their cohort included younger patients with more advanced
disease compared to our patient group. This discrepancy indicate that
age and clinical status are crucial parameters to consider when B cell
subtype data are interpreted.
Th2 cells play a major role in the orchestration of class-switching and
maturation of B cells. We calculated the relationship between the
plasmablasts and Th2 cells and noticed a lower ratio in IgAN which
indicates a relative higher available circulating Th2 count per
plasmablast. However, whether this relationship is valid also at the
lymphoid tissue or whether it has any impact on the actual antibody
production can only be speculated on. Overall, we found no significant
differences in various subsets of T cells in IgAN compared to HC.
We report a higher proportion of long-lived plasma cells (LLPC) in IgAN
patients compared to HC. LLPC contribute to the humoral response by
providing antibody production independent of remaining antibody
producing plasma cells (19). LLPCs consist partly of CD19-negative cells
and reside mostly in bone marrow (19, 20) but can also be found in
inflamed tissue such as in kidneys (20) or the intestinal mucosa (21).
To the best of our knowledge the CD19-negative LLPCs have not previously
been studied in IgAN and it is currently not known in what way this
subpopulation of B cells contributes to the pathological processes. An
interesting finding is that polycystic kidney disease patients also had
an expanded population of CD19-negative LLPC. A plausible explanation
for this finding is not imminent but there is growing interest in how
vascularization and lymphangiogenesis might affect cyst formation and
growth, a process in which B cells are involved (22).
Our data showed an altered monocyte phenotype in patients with IgAN with
an increased proportion of non-classical monocytes. Peripheral monocytes
can be divided into three subgroups, classical (CD14++ CD16),
intermediate (CD14++, CD16+) and non-classical (CD14+, CD16++) monocytes
(23, 24) and our data go in line with a study by Cox and co-workers
(25). A noteworthy difference between our studies is that the patients
had a more preserved renal function in the study by Cox and co-workers.
An interpretation of this observation could be that changes in monocyte
profile appear at an early stage of IgAN disease. In our study increased
levels of non-classical monocytes were accompanied by elevated IL-6. Cox
et al did not analyze IL-6, and therefore we cannot further speculate
whether an expanded population of non-classical monocytes precede an
increase in IL-6.
CD16+ monocytes have previously gained attention and our group has
reported increased CD16+ peripheral monocytes as well as their
accumulation at the site of induced inflammation in patients with CKD
(26). Zhu and co-workers reported a pro-inflammatory role of these cells
in systemic lupus erythematosus (27) and Cols et al reported a potential
role of CD89Low non-classical monocytes in the IgAN
pathogenesis, which together set attention towards this monocyte subset
(17). Of interest is that we found a significant relationship between
MCP-1 levels, a known chemotactic factor for monocytes, and albuminuria
in the current study, which further indicate a role for these cells in
the pathophysiology of IgAN.
We found a significant correlation between sCD40L levels and albuminuria
and higher levels of sCD40L were related to a lower proportion of
pre-switched B cells. This might indicate a role for the CD40-CD40L
pathway in the IgAN pathophysiology, even though the exact in vivo
mechanism of sCD40 is not fully understood. However, sCD40L has been
suggested to act as a suppressor of the immune response by blocking the
CD40-CD40L interaction between B- and T-cells, thereby affecting the B
cell maturation and class-switch and inhibiting immunoglobulin
production (28-30).
In this study we used a method that admits simultaneous analysis of
several immune cells and soluble factors with a minimum of ex vivo
manipulation. Even though the number of subjects is low, the study
includes a well-defined group of IgAN patients, HC and a disease control
group (ADPKD) to validate our results.
In conclusion, we applied an easy-access method to analyze subsets of
immune cells as well as relevant inflammatory mediators in IgAN
patients, healthy controls (HC) and disease controls with ADPKD. Our
data demonstrate an altered B cell profile that indicates a
pathophysiological role of the B cell linage in IgAN and an increased
proportion of non-classical monocytes suggesting their role in the
disease process.