Macrophage tolerance in the gut: It is in the epigenome!

Intestinal macrophages are functionally specialized to phagocytose bacteria, including harmful and invading pathogens, without releasing pro‐inflammatory mediators. This unique functional combination guarantees an optimal defense against millions of gut bacteria while at the same time protecting the host from inflammation‐related tissue damage. IL‐10 seems to be a critical tissue factor involved in this balance and deficiencies in IL‐10 in humans and mice have been shown to deteriorate this fine balance, leading to chronic inflammation and diseases such as Crohn's disease. However, the molecular mechanisms downstream of IL‐10 are poorly understood. In this issue of European Journal of Immunology, Simon et al. [Eur. J. Immunol. 2016. 46: 1912–1925] demonstrate that the chromatin landscape in gut macrophages derived from mice lacking IL‐10 is characteristic of inflammatory macrophages, even in the absence of bacteria. The observed changes of the chromatin landscape represent an important initiating event in chronic intestinal inflammation. These observations are not only an excellent example linking a classical loss‐of‐function experiment to a molecular mechanism responsible for the induction of chronic inflammation; they also open up new avenues for targeting chromatin and remodeling enzymes in Crohn's disease.     

Mucosal IL‐10 and IL‐10 receptor expression patterns in paediatric patients with ulcerative colitis

Interleukin‐10 (IL‐10) is a key anti‐inflammatory cytokine. We aimed to assess IL‐10 and IL‐10 receptor (IL‐10R) expression in the gut, and determine whether these patterns are altered in patients with ulcerative colitis (UC). Formalin‐fixed paraffin‐embedded rectal and transverse colon sections were collected from three groups of patients: (a) control subjects with normal colonoscopy and without history of inflammatory bowel disease; (b) UC patients with extensive colitis or pancolitis (E3/E4 phenotype); and (c) UC patients with limited distal disease (E1/E2 phenotype; n = 8‐10 subjects per group). Immunohistochemistry (IHC) was performed to assess expression patterns of IL‐10, IL‐10R1 and IL‐10R2, and was correlated with clinical, endoscopic and histologic severity indices among patients. A trend towards increased IL‐10 expression was noted in rectal biopsies of patients with active UC, compared with controls. Moreover, IL‐10 levels were significantly increased in transverse colon biopsies of patients with extensive/pancolitis, compared with control subjects and patients with limited distal disease. Rectal IL‐10R1 and IL‐10R2 levels were comparable between control subject and patients with active UC. However, transverse colon IL‐10R1 levels were significantly higher in patients with E3/E4 colitis, compared with controls. Finally, we found no correlation between clinical, endoscopic and histologic severity of inflammation among UC patients and IL‐10, IL‐10R1 or IL‐10R2 expression in rectal sections. Mucosal expression patterns of IL‐10 and IL‐10R, evaluated by IHC, were overall similar between control subjects and patients with active UC. Given IL‐10’s anti‐inflammatory properties, additional studies are required to determine whether signalling through the IL‐10R is altered among these patients.     

Intravenous immunoglobulin (IVIg) or IVIg‐treated macrophages reduce DSS‐induced colitis by inducing macrophage IL‐10 production

Intravenous immunoglobulin (IVIg) is used to treat immune‐mediated diseases but its mechanism of action is poorly understood. We have reported that co‐treatment with IVIg and lipopolysaccharide activates macrophages to produce large amounts of anti‐inflammatory IL‐10 in vitro. Thus, we asked whether IVIg‐treated macrophages or IVIg could reduce intestinal inflammation in mice during dextran sulfate sodium (DSS)‐induced colitis by inducing macrophage IL‐10 production in vivo. Adoptive transfer of IVIg‐treated macrophages reduces intestinal inflammation in mice and collagen accumulation post‐DSS. IVIg treatment also reduces DSS‐induced intestinal inflammation and its activity is dependent on the Fc portion of the antibody. Ex vivo, IVIg induces IL‐10 production and reduces IL‐12/23p40 and IL‐1β production in colon explant cultures. Co‐staining tissues for mRNA, we demonstrate that macrophages are the source of IL‐10 in IVIg‐treated mice; and using IL‐10‐GFP reporter mice, we demonstrate that IVIg induces IL‐10 production by intestinal macrophages. Finally, IVIg‐mediated protection is lost in mice deficient in macrophage IL‐10 production (LysMcre^+/?IL‐10^fl/fl mice). Together, our data demonstrate a novel, in vivo mechanism of action for IVIg. IVIg‐treated macrophages or IVIg could be used to treat people with intestinal inflammation and may be particularly useful for people with inflammatory bowel disease, who are refractory to therapy.     

Downregulation of microRNA‐107 in intestinal CD11c+ myeloid cells in response to microbiota and proinflammatory cytokines increases IL‐23p19 expression

Commensal flora plays an important role in the development of the mucosal immune system and in maintaining intestinal homeostasis. However, the mechanisms involved in regulation of host‐microbiota interaction are still not completely understood. In this study, we examined how microbiota and intestinal inflammatory conditions regulate host microRNA expression and observed lower microRNA‐107 (miR‐107) expression in the inflamed intestines of colitic mice, compared with that in normal control mice. miR‐107 was predominantly reduced in epithelial cells and CD11c⁺ myeloid cells including dendritic cells and macrophages in the inflamed intestines. We demonstrate that IL‐6, IFN‐γ, and TNF‐α downregulated, whereas TGF‐β promoted, miR‐107 expression. In addition, miR‐107 expression was higher in the intestines of germ‐free mice than in mice housed under specific pathogen‐free conditions, and the presence of microbiota downregulated miR‐107 expression in DCs and macrophages in a MyD88‐ and NF‐κB‐dependent manner. We determined that the ectopic expression of miR‐107 specifically repressed the expression of IL‐23p19, a key molecule in innate immune responses to commensal bacteria. We concluded that regulation of miR‐107 by intestinal microbiota and proinflammatory cytokine serve as an important pathway for maintaining intestinal homeostasis.     

Differential signaling of cmvIL‐10 through common variants of the IL‐10 receptor 1

Human IL‐10 (hIL‐10) signaling is mediated by receptors consisting of two subunits, IL‐10 receptor 1 (IL‐10R1) and IL‐10 receptor 2. Two common variants of the IL‐10R1 (Ser 138 Gly (single‐nucleotide polymorphism 3, SNP3) and Gly 330 Arg (SNP4)) are associated with diverse disease phenotypes. Viral homologs to hIL‐10, such as cmvIL‐10, utilize the same IL‐10 receptor complex as part of viral immune evasion strategies. For the present study we hypothesized that IL‐10R1 variants alter the ability of viral IL‐10 to utilize the IL‐10R1 signaling pathway. HeLa cell clones expressing different IL‐10R1 haplotypes (WT or any variant) were incubated with hIL‐10 or cmvIL‐10. In cells expressing IL‐10R1‐WT, cmvIL‐10 (both non‐glycosylated‐ and HeLa‐expressed) resulted in equal or slightly stronger STAT3 phosphorylation compared with hIL‐10. In clones expressing IL‐10R1‐SNP3, IL‐10R1‐SNP4 or IL‐10R1‐SNP3+4, the cmvIL‐10 showed significantly less STAT3 phosphorylation, especially when HeLa‐expressed cytokines were used. Time course experiments demonstrated a slower kinetic of cmvIL‐10 STAT3 activation through the variant IL‐10R1. Similarly, IL‐10R1 variants decreased the cmvIL‐10‐induced SOCS3 and signaling lymphocytic activation molecule mRNA expression. These data suggest that the IL‐10R1 variants differentially reduce the signaling activity of cmvIL‐10 and thereby may affect CMV's ability to escape from the host's immune surveillance.     

Targeted therapy to the IL‐2R using diphtheria toxin and caspase‐3 fusion proteins modulates Treg and ameliorates inflammatory colitis

Pathogenic lymphocytes in the enteric wall of inflammatory bowel disease patients display various abnormalities, including reduced sensitivity to apoptosis. We evaluated a therapeutic approach to elimination of cytotoxic cells, using two IL‐2 fusion proteins, a diphtheria toxin (IL2‐DT) and a caspase‐3 (IL2‐cas) conjugate. In models of acute (dextran sodium sulfate and trinitrobenzene sulfonic acid) and chronic (dextran sodium sulfate) toxic colitis, therapeutic doses of the fusion proteins improved survival and prevented colon shortening. While both chimeric proteins eradicated CD4⁺CD25⁺Foxp3⁺ T cells in mesenteric LN, IL2‐DT caused severe lymphopenia. In contrast, IL2‐cas was equally protective and increased fractional expression of Foxp3. Similar effects of the fusion proteins were observed in healthy mice: IL2‐DT caused lymphopenia and IL2‐cas increased fractional expression of FoxP3. The fusion proteins induced apoptosis in CD25⁺ T cells in vitro, with lower toxicity of IL2‐cas to Foxp3⁺ T cells. These data infer that targeted depletion of cells expressing the IL‐2 receptor has therapeutic potential in models of inflammatory colitis, despite depletion of CD25⁺ Treg. The IL2‐cas fusion protein is particularly relevant to inflammatory bowel disease, as direct internalization of toxic moieties overcomes multiple pathways of resistance to apoptosis of colitogenic T cells.     

CD163 favors Mycobacterium leprae survival and persistence by promoting anti‐inflammatory pathways in lepromatous macrophages

Lepromatous macrophages possess a regulatory phenotype that contributes to the immunosuppression observed in leprosy. CD163, a scavenger receptor that recognizes hemoglobin–haptoglobin complexes, is expressed at higher levels in lepromatous cells, although its functional role in leprosy is not yet established. We herein demonstrate that human lepromatous lesions are microenvironments rich in IDO+CD163+. Cells isolated from these lesions were CD68+IDO+CD163+ while higher levels of sCD163 in lepromatous sera positively correlated with IL‐10 levels and IDO activity. Different Myco‐bacterium leprae (ML) concentrations in healthy monocytes likewise revealed a positive correlation between increased concentrations of the mycobacteria and IDO, CD209, and CD163 expression. The regulatory phenotype in ML‐stimulated monocytes was accompanied by increased TNF, IL‐10, and TGF‐β levels whereas IL‐10 blockade reduced ML‐induced CD163 expression. The CD163 blockade reduced ML uptake in human monocytes. ML uptake was higher in HEK293 cells transfected with the cDNA for CD163 than in untransfected cells. Simultaneously, increased CD163 expression in lepromatous cells seemed to be dependent on ML uptake, and contributed to augmented iron storage in lepromatous macrophages. Altogether, these results suggest that ML‐induced CD163 expression modulates the host cell phenotype to create a favorable environment for myco‐bacterial entry and survival.     

Donor and host B cell‐derived IL‐10 contributes to suppression of graft‐versus‐host disease

Graft‐versus‐host disease (GvHD) is a frequent life‐threatening complication following allogeneic HSC transplantation (HSCT). IL‐10 is a regulatory cytokine with important roles during GvHD, yet its relevant sources, and mode of action, remain incompletely defined in this disease. Using IL‐10‐deficient donor or host mice (BALB/c or C57BL/6, respectively) in a MHC‐mismatched model for acute GvHD, we found a strongly aggravated course of the disease with increased mortality when either donor or host cells could not produce this cytokine. A lack of IL‐10 resulted in increased allogeneic T‐cell responses and enhanced activation of host DCs in spleen and MLNs. Remarkably, IL‐10 was prominently produced by host‐ and donor‐derived CD5^intCD1d^intTIM‐1^int B cells in this disease, and consistent with this, allogeneic HSCT resulted in exacerbated GvHD when mice lacking IL‐10 expression in B cells were used as donor or host, compared with controls. Taken together, this study demonstrates that host and donor B cell‐derived IL‐10 provides a unique mechanism of suppression of acute GvHD, and suggests that DCs are the targets of this B cell‐mediated suppressive effect. These findings open novel therapeutic possibilities based on the use of B cells to increase the feasibility of allogeneic HSCT.     

Activation of murine invariant NKT cells promotes susceptibility to candidiasis by IL‐10 induced modulation of phagocyte antifungal activity

Invariant NKT (iNKT) cells play an important role in a variety of antimicrobial immune responses due to their ability to produce high levels of immune‐modulating cytokines. Here, we investigated the role of iNKT cells in host defense against candidiasis using Jα18‐deficient mice (Jα18^?/?), which lack iNKT cells. Jα18^?/? mice were more resistant to the development of lethal candidiasis than wild‐type (WT) mice. In contrast, treatment of WT mice with the iNKT cell activating ligand α‐galactosylceramide markedly enhanced their mortality after infection with Candida albicans. Serum IL‐10 levels were significantly elevated in WT mice in response to infection with C. albicans. Futhermore, IL‐10 production increased after in vitro coculture of peritoneal macrophages with iNKT cells and C. albicans. The numbers of peritoneal macrophages, the production of IL‐1β and IL‐18, and caspase‐1 activity were also significantly elevated in Jα18^?/? mice after infection with C. albicans. The adoptive transfer of iNKT cells or exogenous administration of IL‐10 into Jα18^?/? reversed susceptibility to candidiasis to the level of WT mice. These results suggest that activation of iNKT cells increases the initial severity of C. albicans infection, most likely mediated by IL‐10 induced modulation of macrophage antifungal activity.     

Reversal of chronic to resolved infection by IL‐10 blockade is LCMV strain dependent

Chronic viral infections lead to CD8⁺ T‐cell exhaustion, characterized by impaired cytokine secretion. The immune‐regulatory cytokine IL‐10 promotes chronicity of infection with lymphocytic choriomeningitis virus (LCMV) Clone 13, as absence of IL‐10 or blocking of IL‐10R during early LCMV Clone 13 infection results in viral clearance. Thus, treatment of humans suffering from chronic viral infections with IL‐10 neutralizing or IL‐10R blocking antibodies was proposed to boost virus‐specific T‐cell responses to enhance control or even clear the viral infection. Here we demonstrate that although CD4⁺ and CD8⁺ T cells can produce elevated levels of cytokines in IL‐10^?/? mice early after infection compared with WT mice, IL‐10^?/? mice cannot clear an infection with the quicker replicating LCMV strain Docile, eventually resulting in T‐cell exhaustion. These data suggest that the success of IL‐10 blockade to control chronic viral infections may critically depend on the virulence of the infecting strain.     

Occupational exposure to trichloroethylene and serum concentrations of IL‐6, IL‐10, and TNF‐alpha

To evaluate the immunotoxicity of trichloroethylene (TCE), we conducted a cross‐sectional molecular epidemiology study in China of workers exposed to TCE. We measured serum levels of IL‐6, IL‐10, and TNF‐α, which play a critical role in regulating various components of the immune system, in 71 exposed workers and 78 unexposed control workers. Repeated personal exposure measurements were taken in workers before blood collection using 3 M organic vapor monitoring badges. Compared to unexposed workers, the serum concentration of IL‐10 in workers exposed to TCE was decreased by 70% (P = 0.001) after adjusting for potential confounders. Further, the magnitude of decline in IL‐10 was >60% and statistically significant in workers exposed to <12 ppm as well as in workers with exposures ≥ 12 ppm of TCE, compared to unexposed workers. No significant differences in levels of IL‐6 or TNF‐α were observed among workers exposed to TCE compared to unexposed controls. Given that IL‐10 plays an important role in immunologic processes, including mediating the Th1/Th2 balance, our findings provide additional evidence that TCE is immunotoxic in humans. Environ. Mol. Mutagen. 54:450–454, 2013. © 2013 Wiley Periodicals, Inc.     

Enhanced protection to Mycobacterium tuberculosis infection in IL‐10‐deficient mice is accompanied by early and enhanced Th1 responses in the lung

IL‐10 regulates the balance of an immune response between pathogen clearance and immunopathology. We show here that Mycobacterium tuberculosis (Mtb) infection in the absence of IL‐10 (IL‐10^?/? mice) results in reduced bacterial loads in the lung. This reduction was preceded by an accelerated and enhanced IFN‐γ response in the lung, an increased influx of CD4⁺ T cells into the lung, and enhanced production of chemokines and cytokines, including CXCL10 and IL‐17, in both the lung and the serum. Neutralization of IL‐17 affected neither the enhanced production of CXCL10 nor the accumulation of IFN‐γ‐producing T cells in the lungs, but led to reduced numbers of granulocytes in the lung and reduced bacterial loads in the spleens of Mtb‐infected mice. This suggests that IL‐17 may contribute to dissemination of Mtb.     

PD‐1 modulates steady‐state and infection‐induced IL‐10 production in vivo

Programmed death‐1 (PD‐1) plays an important role in mediating immune tolerance through mechanisms that remain unclear. Herein, we investigated whether PD‐1 prevents excessive host tissue damage during infection with the protozoan parasite, Toxoplasma gondii. Surprisingly, our results demonstrate that PD‐1‐deficient mice have increased susceptibility to T. gondii, with increased parasite cyst counts along with reduced type‐1 cytokine responses (IL‐12 and IFN‐γ). PD‐1^?/? DCs showed no cell intrinsic defect in IL‐12 production in vitro. Instead, PD‐1 neutralization via genetic or pharmacological approaches resulted in a striking increase in IL‐10 release, which impaired type‐1‐inflammation during infection. Our results indicate that the absence of PD‐1 increases IL‐10 production even in the absence of infection. Although the possibility that such increased IL‐10 protects against autoimmune damage is speculative, our results show that IL‐10 suppresses the development of protective Th1 immune response after T. gondii infection.     

A two-step human culture system replicates intestinal monocyte maturation cascade: Conversion of tissue-like inflammatory monocytes into macrophages

Monocyte maturation program into macrophages (MΦ) is well defined in murine gut under homeostatic or inflammatory conditions. Obviously, in vivo tracking of monocytes in inflamed tissues remains difficult in humans. Furthermore, in vitro models fall short in generating the surrogates of transient extravasated tissue inflammatory monocytes. Here, we aimed to unravel environmental cues that replicated the human monocyte “waterfall” process in vitro by first, generating tissue-like inflammatory monocytes, which were then shifted toward MΦ. Purified CD14⁺CD16⁻ monocytes, cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN-γ and IL23, differentiated into CD14⁺CD163⁻ cells that displayed a monocyte-like morphology. In vitro generated inflammatory CD14⁺CD163⁻ (inflammatory monocyte-like cells) cells promoted IL-1β-dependent memory Th17 and Th17/Th1 responses, like the CD14⁺CD163⁻ mo-like cells that accumulate in inflamed colon of Crohn's disease patients. Next, in vitro generated inflammatory monocyte-like cells converted to functional CD163⁺ MΦ following exposure to TGF-β and IL10. Gene set enrichment analysis further revealed a shared molecular signature between converted CD163⁺ MΦ and MΦ detected in various inflamed nonlymphoid and lymphoid diseased tissues. Our findings propose a two-step in vitro culture that recapitulates human monocyte maturation cascade in inflamed tissue. Manipulation of this process might open therapeutic avenues for chronic inflammatory disorders.     

Human gingiva‐derived mesenchymal stem cells alleviate inflammatory bowel disease via IL‐10 signalling‐dependent modulation of immune cells

Current evidence indicates that inflammatory bowel disease (IBD) is caused primarily by impaired mucosal immunity, resulting in an imbalance between epithelial barrier function and tissue inflammation. Human gingiva‐derived mesenchymal stem cells (GMSCs) exhibit immunomodulatory and anti‐inflammatory effects in a variety of immunity‐ and inflammation‐associated diseases. However, the role of GMSCs in treating IBD has not been elucidated. Our study, therefore, examined the therapeutic effect and mechanism of GMSCs in a murine colitis model of IBD. Our results indicate that the infusion of GMSCs significantly prolonged survival and relieved symptoms. Phenotype analyses showed that the frequencies of NK1.1⁺ and CD11b⁺ cells, as well as CD4 T cells in the spleen, were suppressed in GMSC‐treated mice compared with the PBS‐ or fibroblast‐treated control groups. Additionally, GMSC treatment markedly increased the numbers of interleukin (IL)‐10⁺ regulatory T cells, reduced the secretion of pro‐inflammatory cytokines, and increased production of anti‐inflammatory cytokines. A mechanistic study revealed that anti‐IL‐10R antibody abolished the protective effect of GMSCs compared with mice treated with anti‐IgG antibody. Thus, our results indicate that GMSCs play a critical role in alleviating colitis by modulating inflammatory immune cells via IL‐10 signalling.     

Understanding the molecular mechanisms of the multifaceted IL‐10‐mediated anti‐inflammatory response: Lessons from neutrophils

Analysis of the molecular mechanisms governing the ability of IL‐10 to keep inflammation under control has highlighted the existence of a great degree of plasticity and specificity with regard to innate immune cells. In this respect, neutrophils represent a perfect example of innate immune cells conditioned by external signals (for instance, by LPS), as well as by intracellular regulatory pathways, that render them optimally responsive to IL‐10 only when required. The focus of this review are the recent experimental findings that have uncovered the sophisticated and complex molecular mechanisms responsible for the modulation of pro‐ and anti‐inflammatory cytokine production by IL‐10 in neutrophils and other innate immune cells. Understanding how IL‐10 exerts its anti‐inflammatory response, particularly in the case of neutrophils, will provide novel clues leading, hopefully, to the therapeutic control of neutrophil‐driven inflammatory reactions, such as septic infections, rheumatoid arthritis, osteoarthritis and chronic obstructive pulmonary disease.     

IL‐10 differentially controls the infiltration of inflammatory macrophages and antigen‐presenting cells during inflammation

The inflammatory activation and recruitment of defined myeloid populations is essential for controlling the bridge between innate and adaptive immunity and shaping the immune response to microbial challenge. However, these cells exhibit significant functional heterogeneity and the inflammatory signals that differentially influence their effector characteristics are poorly characterized. In this study, we defined the phenotype of discrete subsets of effective antigen‐presenting cells (APCs) in the peritoneal cavity during peritonitis. When the functional properties of these cells were compared to inflammatory monocyte‐derived macrophages we noted differential responses to the immune‐modulatory cytokine IL‐10. In contrast to the suppressive actions of IL‐10 on inflammatory macrophages, the recruitment of APCs was relatively refractory and we found no evidence for selective inhibition of APC differentiation. This differential response of myeloid cell subsets to IL‐10 may thus have limited impact on development of potentially tissue‐damaging adaptive immune responses, while restricting the magnitude of the inflammatory response. These findings may have clinical relevance in the context of peritoneal dialysis patients, where recurrent infections are associated with immune‐mediated membrane dysfunction, treatment failure, and increased morbidity.