Short Communication - Journal of Cellular Immunology (2021) Volume 3, Issue 2
IL-10 Responsiveness and Anti-TNF Therapy in Inflammatory Bowel Disease
Felicia M. Bloemendaal1,2, Charlotte P. Peters1, Anje A. te Velde1,2, Cyriel Y. Ponsioen1, Gijs R. van den Brink1,2,3, Manon E. Wildenberg1,2#, Pim J. Koelink2#*
1Department of Gastroenterology and Hepatology, University of Amsterdam, Amsterdam, the Netherlands
2Tytgat Institute for Liver and Intestinal Research, Amsterdam UMC, location AMC, AG&M Amsterdam, University of Amsterdam, Amsterdam, the Netherlands
3Current address: Roche Innovation Center Basel, F. Hoffmann-La Roche AG, Basel, Switzerland
- *Corresponding Author:
- Pim J. Koelink
Received date: December 05, 2020; Accepted date: January 28, 2021
Citation: Bloemendaal FM, Peters CP, te Velde AA, Ponsioen CY, van den Brink GR, Wildenberg ME, et al. IL-10 Responsiveness and
Anti-TNF Therapy in Inflammatory Bowel Disease. J Cell Immunol. 2021; 3(2): 91-96.
Copyright: © 2021 Bloemendaal FM, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
IBD, IL-10, Anti-TNF, Macrophages, Colitis
Macrophages, IL-10 and Anti-TNF Therapy in IBD
Inflammatory bowel disease (IBD) is a multifactorial disease in which both genetic and environmental factors play an important role, although the precise cause remains obscure. It is clear that both the innate and adaptive immunity are involved in acquiring mucosal immune homeostasis in the intestine, which is dysregulated in IBD.
Interleukin-10 & IBD
Several studies have pinpointed Interleukin(IL)-10 signaling as a key player in the maintenance of intestinal mucosal immune homeostasis. IL-10 is a potent antiinflammatory cytokine that is produced by a wide variety of different cell types, like T-cells, macrophages, dendritic cells and epithelial cells . Most hematopoietic cells can respond to IL-10 via expression of the IL-10 receptor (IL- 10 R), which is a heterodimer composed of IL-10 Rα and IL-10 Rβ . Macrophages are among the cells that express the highest level of IL-10 R. Deleterious mutations in IL-10 or IL-10 R cause severe early onset IBD . This situation is recapitulated in mice where deficiencies in either IL-10 or IL-10 R cause spontaneous colitis [4,5]. Also in genomewide association studies, single-nucleotide polymorphisms (SNPs) in the IL-10 pathway have been linked to the risk of developing IBD .
While initially it was assumed that IL-10 suppressed inflammatory responses by directly affecting T-cells , further experimental studies pointed out a pivotal role for IL-10 signaling within the myeloid compartment as the absence of IL-10 Rα in intestinal macrophages resulted in spontaneous colitis [8,9]. Therefore, IL-10 signaling within macrophages plays a central role in the regulation of intestinal mucosal homeostasis.
Recombinant IL-10 as a therapeutic strategy has been tested in various forms. Thus far, efforts to treat IBD patients with recombinant IL-10 systemically have not shown convincing evidence of therapeutic efficacy [10-13]. However, against the background of recent evidence that the primary issue in IBD seems to be a defect in IL-10 sensing rather than availability, administration of IL-10 is less likely to be effective, unless strategies to increase responsiveness will be developed.
Macrophages & IBD
Macrophages are part of the myeloid compartment and can orchestrate all stages of the immune response: 1) as a first line of defense against bacteria, 2) in the resolution of inflammation, and finally 3) in wound healing and tissue repair . Macrophages are plastic cells that can adapt their phenotype to environmental stimuli. Macrophages have classically been subdivided into either a pro-inflammatory (M1) or a regulatory (M2) subtype, but it is now generally accepted that macrophages can adopt a spectrum of phenotypes in vivo as many different stimuli can influence their behavior . In contrast to other organs, where
tissue-resident macrophages are derived from the yolk-sac
during embryogenesis, most macrophages in the intestine
are constantly replenished by an influx of bone marrow
derived monocytes . In the intestine, macrophages
are continuously exposed to microbial components that
can potentially evoke an immune response. To prevent
constant inflammation, they possess the capacity to clear
pathogens and scavenge cell debris without further alerting
other players of the immune cascade. This tolerogenic state
is marked by a diminished production of inflammatory
factors like nitric oxide, reactive oxygen species and proinflammatory
cytokines, despite the presence of microbial
stimuli [17,18]. This anergic state is thought to be actively
maintained via inhibitory molecules like IL-10 . Thus,
while retaining their phagocytic and bactericidal capacity,
intestinal macrophages play a crucial role in preventing
an excessive immune response against the microbiome. In
active IBD the balance between these seemingly opposing
tasks is disrupted, leading to an excessive inflammatory
cascade. Single-cell analysis confirmed the abundance of
an inflammatory macrophage subset in active ileal Crohn’s
disease (CD) compared to uninflamed lesions . This
subset was characterized by an increased expression of
nuclear factor κB (NF-κB) and inflammatory molecules
like IL-1, IL-6, IL-23, TNFα and Oncostatin M (OSM). A
similar study for colonic CD showed that inflammatory
CD14+CD64hi CD163-/dim IL-23 producing macrophages were enriched in inflamed lesions compared to uninflamed lesions . Thus, during active disease the larger
proportion of CD macrophages displays an inflammatory
phenotype while during disease remission regulatory
subsets prevail. Therefore, the interest in reprogramming
macrophages to resolve inflammation in a therapeutic
setting in IBD has grown over the last years.
Anti-TNF therapy in IBD
Anti-Tumor Necrosis Factor-(TNF)-α monoclonal antibodies (mAbs) are considered a breakthrough in the therapeutic treatment of IBD in clinical practice. Interestingly, a range of clinical observations has shown that only the full mAbs against TNF, adalumimab and infliximab, have a substantial therapeutic effect in IBD, in contrast to other anti-TNF agents like certolizumab (pegylated Fab’ fragment), onercept (soluble TNF receptor I) and etanercept (TNF receptor II-Fc fusion protein) . While these TNF blocking strategies established their value in several immune mediated diseases like Rheumatoid Arthritis (RA), only the full mAbs demonstrated induction of complete endoscopic remission in IBD. This suggests that additional features besides TNF neutralization are necessary for therapeutic efficacy and for the induction of mucosal healing in IBD. The Fc region of an antibody can bind to Fcγ receptors (FcγR) which are highly expressed by myeloid cells. Over the last decade we have found strong evidence for FcγR dependent macrophage modulation as a core mechanism of action for the efficacy of anti-TNF mAbs in IBD. The hypothesis that the Fc region might play a role in the therapeutic effect of anti-TNFs originated from unsuccessful experiments to find direct effects of anti-TNFs on T-cells, in particular induction of apoptosis . It wasn’t until we used a mixed lymphocyte reaction
(MLR), created by mixing peripheral blood mononuclear
cells (PBMCs) of two different donors, where monocytes
of one donor activate the T-cells of the other donor, that
we found an effect of anti-TNFs. In this set-up only the
anti-TNF mAbs (infliximab, adalimumab and golimumab)
inhibited T-cell proliferation, in contrast to other TNF
blocking agents like etanercept or certolizumab. The
effect was completely dependent on the Fc-mediated
polarization of the monocytes in the MLR to an ‘M2-
like’ regulatory macrophage phenotype that suppressed
T-cell proliferation . We were subsequently able to reproduce the dependence on the Fc region in preclinical experiments in vivo. First, we showed that an anti-TNF
mAb with high Fc binding affinity was effective in reducing
colitis in contrast to an anti-TNF mAb with a low Fc binding
affinity in an experimental IBD mouse model, while both
mAbs were equally effective in an experimental model of
RA . This showed that Fc interaction was necessary for
therapeutic efficacy in the IBD model but not in the model
for RA, where reducing arthritis seemed solely dependent
on TNF blockade. We found that mice lacking activating
FcγR were completely unresponsive to anti-TNF mAb
therapy in the same IBD model, while an afucosylated
anti-TNF mAb with increased affinity for FcγR showed
increased efficacy . In both mice and humans Fc
engagement by anti-TNF promoted the formation of
regulatory macrophages. Characterization of the intestinal
myeloid compartment of mice with active colitis showed
an increase in CD206+ regulatory macrophages after anti-
TNF treatment compared to treatment with an isotype
control antibody. The number of CD206+ macrophages
was even higher with an afucosylated anti-TNF and,
conversely, percentages remained low in anti-TNF treated
mice that were deficient for activating FcγR . Likewise,
the number of CD206+ macrophages increased in the
lamina propria of IBD patients responding to anti-TNF
mAb therapy. In contrast, this was not the case in anti-
TNF non-responders .
Further characterization of regulatory features showed that anti-TNF mAbs increased the ratio of IL- 10 versus IL-12/IL-23p40 production in inflammatory macrophages differentiated in vitro . Similarly, anti- TNF mAbs suppressed IL-12/IL-23 p40 in lamina propria macrophages isolated from IBD patients with active disease.
Overall, these data strongly suggest that Fc engagement of activating Fc receptors polarizes intestinal macrophages to an ‘M2-like’ regulatory phenotype and that this is required for therapeutic efficacy.
Anti-TNF therapy effectiveness depends on IL-10
signaling in macrophages in IBD
In our recent paper regarding anti-TNF in IBD we show that IL-10 signaling in macrophages is necessary for CD206+ macrophage induction and for response to anti- TNF mAbs in experimental colitis. Several reports describe patients with germline mutations in the IL-10 pathway to be unresponsive to anti-TNF [3,28]. Interestingly,
although IL-10 deficient mice were refractory to anti-TNF therapy as shown previously , they did respond to
antibodies against IL-12/IL-23p40. Perhaps macrophages
become refractory to Fc engagement when IL-10 signaling
is disrupted, locking them in an IL-12/IL-23 secreting
inflammatory state that can only be blocked further
A study in pediatric CD patients showed that monocytes of patients were less responsive to IL-10 compared to healthy controls presumably due to the reduced expression of IL- 10 Rα . We confirmed this finding in adult CD patients. We found that the expression of IL-10 Rα was lower in CD14+ monocytes of CD patients compared to healthy controls in peripheral blood obtained before the start of anti-TNF therapy (Figure 1A). Indeed, by performing flow cytometry for pSTAT3 in combination with CD14 in
PBMCs with or without IL-10 stimulation, we also found a
decreased IL-10 responsiveness in the monocytes of these
CD patients compared to healthy controls (Figure 1B).
Although the patient numbers were low, we found that
both the pretreatment responsiveness to IL-10 and IL10
Rα expression levels of CD14+ monocytes from anti-TNF
non-responding CD patients were significantly decreased
compared to healthy controls, which was not the case for
anti-TNF responsive CD patients (Figure 1C/D), indicating
this could potentially be used as a predictive marker for anti-
TNF response. Future research could focus on unravelling
mechanisms that maintain IL-10 unresponsiveness.
Materials & Methods
CD patient study
CD patients with complaints of active disease, scheduled for colonoscopy to determine disease activity, were asked to participate with written informed consent before new anti- TNF remission therapy was initiated. Endoscopic response to induction therapy was determined by endoscopy after 8 weeks. During colonoscopy the Crohn’s Disease Endoscopic Index of Severity (CDEIS) was determined and response was defined as >50 % reduction in CDEIS after 8 weeks of induction therapy with either adalumimab or infliximab. Two patients turned out to be therapy intolerant and therefore no response was determined. For patient information see Table 1. Prior to treatment
initiation a blood sample was taken, and PBMCs were
isolated by Ficoll Paque density-gradient centrifugation.
After cell isolation, samples were cryopreserved with
DMSO using Mr Frosty (Thermo Fisher Scientific,
Waltham, Massachusetts, USA) and afterwards stored
in liquid nitrogen. Healthy volunteers were also asked
to participate with written informed consent. The study
protocol was approved by the Medical Ethical Committee
of the AMC (Amsterdam UMC, METC09/113#09.17.0938)
| Patient|| Gender|| Age at inclusion (years)|| CDEIS (start)|| CDEIS (8 weeks)|| CRP (start)|| CRP (8 weeks)|| Montreal classification*|| anti-TNF|
| A1/A2/A3|| L1/L2/L3/L4|| B1/B2/B3/+p|
CDEIS: Crohn’s Disease Endoscopic Index of Severity; CRP: C-reactive Protein (mg/L);
* Montreal classification: Age at diagnosis: A1: <17 years, A2: 17–40 years, A3: >40 years; Maximal location of disease: L1: ileal, L2: colonic, L3: ileocolonic, L4: upper disease; Maximal disease behaviour: B1: non-stricturing, non-penetrating, B2: stricturing, B3: penetrating, p: perianal disease, IFX: Infliximab, ADA: Adalimumab
Table 1: Patient characteristics.
PBMCs were thawed and washed with prewarmed RPMI
supplemented with 10% heat-inactivated FCS. After centrifugation @ 500 x g cell pellets were resuspended
in 400 μl of medium and divided over two separate tubes
for flow cytometric analyses of IL-10 Rα expression or
pSTAT3 induction. For the IL-10 Rα cells were blocked
with Fc block (1:20, BD Biosciences, 564220) in 0.5%
BSA/PBS for 30 minutes at room temperature (RT) and
subsequently stained with either CD14-PE-Cy7 (1:100,
Clone 61D3, eBiosciences) alone (Fluorescence Minus
One (FMO)) or in combination with IL-10-Rα-PE(1:20,
clone REA239, Miltenyi Biotechnology). The mean
fluorescence intensity (MFI) of IL10Rα was determined
by subtracting the background MFI of unstained gated
CD14+ cells (FMO). Other cells were either stimulated
with 10 ng/ml rhIL10 (R&D, 217-IL-025) for 15 minutes
or left untreated at 37°C. Afterwards cells were fixated by
adding BD Cytofix/Cytoperm (BD Biosciences, 554722)
in a 1:1 ratio and left for 30 min @ RT in the dark. After
centrifugation cells were permeabilized using ice cold
methanol for 30 min @ 4°C. After thorough washings
with 0.5 % BSA/PBS cells were subsequently stained with
CD14-PE-Cy7 and pSTAT3-APC (1:50, clone LUVNKLA,
eBiosciences). Induction of pSTAT3 was calculated by
dividing the IL-10-stimulated pSTAT3 MFI of the CD14+
gated population by the pSTAT3 MFI of the unstimulated
CD14+ gated population. Flow cytometry was performed
using a FACS Fortessa (BD) and FlowJo software (Treestar
Inc., Ashland, OR) was used for analysis.
Conflict of Interest
The authors have no conflicts of interest related to this
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