Macrophages are important innate immune cells that are associated with two distinct phenotypes: a pro-inflammatory (or classically activated) subset with prototypic macrophage functions such as inflammatory cytokine production and bactericidal activity, and an anti-inflammatory (or alternatively activated (AAM)) subset linked with wound healing and tissue repair processes. In this study, we examined the effect of interlukein-6 on human and murine macrophage polarization. The results indicate that despite being commonly associated with pro-inflammatory functions and being implicated in the pathogenesis/pathophysiology of numerous inflammatory diseases, interleukin-6 can enhance the polarization of AAMs, based on increased expression of hallmark markers: arginase-1, Ym1 and CD206; this effect required the AAM differentiating cytokines, IL-4 and IL-13. Co-treatment of AAMs with IL-6 resulted in spontaneous release of IL-10, suppressed LPS-induced nitric oxide production and inhibited cytokine production by activated CD4+ T cells – immunoregulatory features not observed in the ‘parent’ IL-4+IL-13-induced AAM. The effect of IL-6 required signal transducer and activator of transcription (STAT)-3, was partially dependent on up-regulation of the IL4Rα chain, and was independent of autocrine IL-10. In the presence of IFNγ, IL-6 promoted the production of IL-1β and TNFα suggesting that this cytokine can enhance the phenotype to which a macrophage has committed. This finding may explain the pleiotrophic nature of IL-6, where it is associated with the perpetuation and enhancement of disease in inflammatory situations, but is also necessary for resolution of inflammation and adequate wound healing to occur in others. Thus, the potential benefit of IL-6 in promoting an AAM, with its’ anti-inflammatory and wound healing ability, may need to be considered in immunotherapies aimed at in vivo modulation or inhibition of IL-6.

Epithelial permeability is often increased in inflammatory bowel diseases. We hypothesized that perturbed mitochondrial function would cause barrier dysfunction and hence epithelial mitochondria could be targeted to treat intestinal inflammation. Mitochondrial dysfunction was induced in human colon-derived epithelial cell lines or colonic biopsy specimens using dinitrophenol, and barrier function was assessed by transepithelial flux of Escherichia coli with or without mitochondria-targeted antioxidant (MTA) cotreatment. The impact of mitochondria-targeted antioxidants on gut permeability and dextran sodium sulfate (DSS)-induced colitis in mice was tested. Mitochondrial superoxide evoked by dinitrophenol elicited significant internalization and translocation of E. coli across epithelia and control colonic biopsy specimens, which was more striking in Crohn's disease biopsy specimens; the mitochondria-targeted antioxidant, MitoTEMPO, inhibited these barrier defects. Increased gut permeability and reduced epithelial mitochondrial voltage-dependent anion channel expression were observed 3 days after DSS. These changes and the severity of DSS-colitis were reduced by MitoTEMPO treatment. In vitro DSS-stimulated IL-8 production by epithelia was reduced by MitoTEMPO. Metabolic stress evokes significant penetration of commensal bacteria across the epithelium, which is mediated by mitochondria-derived superoxide acting as a signaling, not a cytotoxic, molecule. MitoTEMPO inhibited this barrier dysfunction and suppressed colitis in DSS-colitis, likely via enhancing barrier function and inhibiting proinflammatory cytokine production. These novel findings support consideration of MTAs in the maintenance of epithelial barrier function and the management of inflammatory bowel diseases.

Alternatively activated macrophages (AAMs) (or M2a) can inhibit colitis but may also be associated with fibrosis. Thus, by using the dinitrobenzene sulfonic (DNBS) murine model of colitis, this study aimed to determine whether 1) bone marrow (BM)-derived AAMs could reduce colitis, 2) any anticolitic effect of BM-AAMs was IL-10 dependent, and 3) repeated AAM treatments remained effective and were associated with fibrosis in the gut or other tissues. Balb/c mice received AAMs (10(6) intraperitoneally) from wild-type (WT) or IL-10(-/-) mice 48 h prior to DNBS (3 mg intrarectally) with disease assessed 72 h later, or they received three doses of DNBS at 2-wk intervals ± AAMs 6 h post-DNBS to mimic a treatment regimen. DNBS-treated mice developed colitis; this was significantly less severe in mice receiving WT AAMs and less so in animals given IL-10(-/-) AAMs, indicating a role for IL-10 in the inhibition of DNBS-driven colitis. Similarly, after the third AAM treatment lesser colonic histopathology was observed compared with time-matched DNBS-only-treated animals, and notably there was no evidence of increased fibroses in the colon, terminal ileum, lung, or liver of AAM-treated mice as assessed by quantitative PCR for prolyl-4-hydrolase, α-smooth muscle actin, and collagen (type IIIα) and histochemical and biochemical assessment of collagen deposition. This study provides mechanistic insight to the anticolitic capacity of AAMs and indicates that repeated adoptive transfer of ex vivo programmed BM-AAMs is safe and efficacious in the treatment of DNBS-induced murine colitis, providing additional support for their consideration as an immunotherapy.

The short-chain fatty acid butyrate is produced by fermentation of dietary fiber by the intestinal microbiota; butyrate is the primary energy source of colonocytes and has immunomodulatory effects. Having shown that macrophages differentiated with IL-4 [M(IL-4)s] can suppress colitis, we hypothesized that butyrate would reinforce an M(IL-4) phenotype. Here, we show that in the presence of butyrate M(IL-4)s display reduced expression of their hallmark markers Arg1 and Ym1 and significantly suppressed LPS-induced nitric oxide, IL-12p40, and IL-10 production. Butyrate treatment likely altered the M(IL-4) phenotype via inhibition of histone deacetylation. Functionally, M(IL-4)s treated with butyrate showed increased phagocytosis and killing of bacteria, compared with M(IL-4) and this was not accompanied by enhanced proinflammatory cytokine production. Culture of regulatory T cells with M(IL-4)s and M(IL-4 + butyrate)s revealed that both macrophage subsets suppressed expression of the regulatory T-cell marker Foxp3. However, Tregs cocultured with M(IL-4 + butyrate) produced less IL-17A than Tregs cocultured with M(IL-4). These data illustrate the importance of butyrate, a microbial-derived metabolite, in the regulation of gut immunity: the demonstration that butyrate promotes phagocytosis in M(IL-4)s that can limit T-cell production of IL-17A reveals novel aspects of bacterial-host interaction in the regulation of intestinal homeostasis.

Helminth parasites provoke multicellular immune responses in their hosts that can suppress concomitant disease. The gut lumen-dwelling tapeworm Hymenolepis diminuta, unlike other parasites assessed as helminth therapy, causes no host tissue damage while potently suppressing murine colitis. With the goal of harnessing the immunomodulatory capacity of infection with H. diminuta, we assessed the putative generation of anti-colitic regulatory B cells following H. diminuta infection. Splenic CD19+ B cells isolated from mice infected 7 [HdBc(7d)] and 14 d (but not 3 d) previously with H. diminuta and transferred to naive mice significantly reduced the severity of dinitrobenzene sulfonic acid (DNBS)-, oxazolone-, and dextran-sodium sulfate–induced colitis. Mechanistic studies with the DNBS model, revealed the anti-colitic HdBc(7d) was within the follicular B cell population and its phenotype was not dependent on IL-4 or IL-10. The HdBc(7d) were not characterized by increased expression of CD1d, CD5, CD23, or IL-10 production, but did spontaneously, and upon LPS plus anti-CD40 stimulation, produce more TGF-β than CD19+ B cells from controls. DNBS-induced colitis in RAG1−/− mice was inhibited by administration of HdBc(7d), indicating a lack of a requirement for T and B cells in the recipient; however, depletion of macrophages in recipient mice abrogated the anti-colitic effect of HdBc(7d). Thus, in response to H. diminuta, a putatively unique splenic CD19+ B cell with a functional immunoregulatory program is generated that promotes the suppression of colitis dominated by TH1, TH2, or TH1-plus-TH2 events, and may do so via the synthesis of TGF-β and the generation of, or cooperation with, a regulatory macrophage.

Interleukin (IL)-22, an immune cell-derived cytokine whose receptor expression is restricted to non-immune cells (e.g. epithelial cells), can be anti-inflammatory and pro-inflammatory. Mice infected with the tapeworm Hymenolepis diminuta are protected from dinitrobenzene sulphonic acid (DNBS)-induced colitis. Here we assessed expulsion of H. diminuta, the concomitant immune response and the outcome of DNBS-induced colitis in wild-type (WT) and IL-22 deficient mice (IL-22-/-) ± infection. Interleukin-22-/- mice had a mildly impaired ability to expel the worm and this correlated with reduced or delayed induction of TH2 immunity as measured by splenic and mesenteric lymph node production of IL-4, IL-5 and IL-13 and intestinal Muc-2 mRNA and goblet cell hyperplasia; in contrast, IL-25 increased in the small intestine of IL-22-/- mice 8 and 12 days post-infection compared to WT mice. In vitro experiments revealed that H. diminuta directly evoked epithelial production of IL-25 that was inhibited by recombinant IL-22. Also, IL-10 and markers of regulatory T cells were increased in IL-22-/- mice that displayed less DNBS (3 mg, ir. 72h)-induced colitis. Wild-type mice infected with H. diminuta were protected from colitis, as were infected IL-22-/- mice and the latter to a degree that they were almost indistinguishable from control, non-DNBS treated mice. Finally, treatment with anti-IL-25 antibodies exaggerated DNBS-induced colitis in IL-22-/- mice and blocked the anti-colitic effect of infection with H. diminuta. Thus, IL-22 is identified as an endogenous brake on helminth-elicited TH2 immunity, reducing the efficacy of expulsion of H. diminuta and limiting the effectiveness of the anti-colitic events mobilized following infection with H. diminuta in a non-permissive host.

BACKGROUND AND PURPOSEAlternatively activated macrophages (AAMs) are important cells in the resolution of inflammation and tissue repair. We examined the impact of myofibroblasts, a vital cell in wound healing and tissue repair, on the development and function of AAMs. EXPERIMENTAL APPROACHThe interaction between AAMs and myofibroblasts was tested using conditioned medium from murine dermal myofibroblasts and bone marrow-derived macrophages. AAMs were differentiated with IL-4 and IL-13. KEY RESULTSConditioned medium from myofibroblasts enhanced the expression of AAM markers, arginase 1 and Ym1 (chitinase-3-like 3) and the spontaneous production of IL-10, while suppressing LPS-induced nitric oxide production. IL-6 from the myofibroblasts contributed to the amplification of the AAM phenotype; the selective COX-2 inhibitor, NS-398, significantly reduced the ability of myofibroblasts to promote an AAM phenotype. Pharmacological analyses indicated that myofibroblast-derived IL-6 enhanced arginase activity and spontaneous IL-10 output, while PGE 2 , via the EP 4 receptor, enhanced arginase expression and LPS-evoked IL-10 production. PGD 2 suppressed LPS-evoked nitric oxide via the DP 1 receptor. Reciprocally, conditioned medium from macrophages treated with IL-4 + IL-13 and myofibroblast conditioned medium components, but not macrophages given IL-4 + IL-13 only, reduced myofibroblast migration, the expression of COX-2, and the production of PGE 2 and PGD 2 . CONCLUSIONS AND IMPLICATIONSThese findings define mechanisms by which myofibroblasts enhance an AAM phenotype, which can promote wound healing directly, and/or via feedback communication to the myofibroblast, subsequently down-regulating its capacity to promote AAM function. This is an important homeostatic regulatory pathway in wound healing that can also limit unwanted fibrosis.Abbreviations AAM, alternatively activated macrophage; ECM, extracellular matrix; MCM, macrophage conditioned medium; MFbCM, myofibroblast conditioned medium; M-CSF, macrophage-colony stimulating factor; SMA, smooth muscle actin BJP British Journal of Pharmacology

Substantial data show that infection with helminth parasites ameliorates colitis; however, oxazolone-induced colitis is exaggerated in mice infected with the tapeworm, Hymenolepis diminuta. We tested the hypothesis that the IL-5 response to helminth infection enhances the severity of oxazolone-induced colitis.