Interleukin-4 Programmed Macrophages Suppress Colitis and Do Not Enhance Infectious-Colitis, Inflammation-Associated Colon Cancer or Airway Hypersensitivity.

The murine interleukin-4 treated macrophage (MIL4) exerts anti-inflammatory and pro-healing effects and has been shown to reduce the severity of chemical-induced colitis. Positing M(IL4) transfer as an anti-inflammatory therapy, the possibility of side-effects must be considered. Consequently, bone marrow-derived M(IL4)s were administered via intraperitoneal injection to mice concomitant with Citrobacter rodentium infection (infections colitis), azoxymethane/dextran sodium sulphate (AOM/DSS) treatment [a model of colorectal cancer (CRC)], or ovalbumin sensitization (airway inflammation). The impact of M(IL4) treatment on C. rodentium infectivity, colon histopathology, tumor number and size and tissue-specific inflammation was examined in these models. The anti-colitic effect of the M(IL4)s were confirmed in the di-nitrobenzene sulphonic acid model of colitis and the lumen-to-blood movement of 4kDa FITC-dextran and bacterial translocation to the spleen and liver was also improved by M(IL4) treatment. Analysis of the other models of disease, that represent comorbidities that can occur in human inflammatory bowel disease (IBD), revealed that M(IL4) treatment did not exaggerate the severity of any of the conditions. Rather, there was reduction in the size (but not number) of polyps in the colon of AOM/DSS-mice and reduced infectivity and inflammation in C. rodentium-infected mice in M(IL4)-treated mice. Thus, while any new therapy can have unforeseen side effects, our data confirm and extend the anti-colitic capacity of murine M(IL4)s and indicate that systemic delivery of one million M(IL4)s did not exaggerate disease in models of colonic or airways inflammation or colonic tumorigenesis.

Cooperation between host immunity and the gut bacteria is essential for helminth-evoked suppression of colitis.

BACKGROUND: Studies on the inhibition of inflammation by infection with helminth parasites have, until recently, overlooked a key determinant of health: the gut microbiota. Infection with helminths evokes changes in the composition of their host's microbiota: one outcome of which is an altered metabolome (e.g., levels of short-chain fatty acids (SCFAs)) in the gut lumen. The functional implications of helminth-evoked changes in the enteric microbiome (composition and metabolites) are poorly understood and are explored with respect to controlling enteric inflammation. METHODS: Antibiotic-treated wild-type, germ-free (GF) and free fatty-acid receptor-2 (ffar2) deficient mice were infected with the tapeworm Hymenolepis diminuta, then challenged with DNBS-colitis and disease severity and gut expression of the il-10 receptor-α and SCFA receptors/transporters assessed 3 days later. Gut bacteria composition was assessed by 16 s rRNA sequencing and SCFAs were measured. Other studies assessed the ability of feces or a bacteria-free fecal filtrate from H. diminuta-infected mice to inhibit colitis. RESULTS: Protection against disease by infection with H. diminuta was abrogated by antibiotic treatment and was not observed in GF-mice. Bacterial community profiling revealed an increase in variants belonging to the families Lachnospiraceae and Clostridium cluster XIVa in mice 8 days post-infection with H. diminuta, and the transfer of feces from these mice suppressed DNBS-colitis in GF-mice. Mice treated with a bacteria-free filtrate of feces from H. diminuta-infected mice were protected from DNBS-colitis. Metabolomic analysis revealed increased acetate and butyrate (both or which can reduce colitis) in feces from H. diminuta-infected mice, but not from antibiotic-treated H. diminuta-infected mice. H. diminuta-induced protection against DNBS-colitis was not observed in ffar2-/- mice. Immunologically, anti-il-10 antibodies inhibited the anti-colitic effect of H. diminuta-infection. Analyses of epithelial cell lines, colonoids, and colon segments uncovered reciprocity between butyrate and il-10 in the induction of the il-10-receptor and butyrate transporters. CONCLUSION: Having defined a feed-forward signaling loop between il-10 and butyrate following infection with H. diminuta, this study identifies the gut microbiome as a critical component of the anti-colitic effect of this helminth therapy. We suggest that any intention-to-treat with helminth therapy should be based on the characterization of the patient's immunological and microbiological response to the helminth.

STAT1 Is Required for Decreasing Accumulation of Granulocytic Cells via IL-17 during Initial Steps of Colitis-Associated Cancer.

Signal transducer and activator of transcription 1 (STAT1) acts as a tumor suppressor molecule in colitis-associated colorectal cancer (CAC), particularly during the very early stages, modulating immune responses and controlling mechanisms such as apoptosis and cell proliferation. Previously, using an experimental model of CAC, we reported increased intestinal cell proliferation and faster tumor development, which were consistent with more signs of disease and damage, and reduced survival in STAT1-/- mice, compared with WT counterparts. However, the mechanisms through which STAT1 might prevent colorectal cancer progression preceded by chronic inflammation are still unclear. Here, we demonstrate that increased tumorigenicity related to STAT1 deficiency could be suppressed by IL-17 neutralization. The blockade of IL-17 in STAT1-/- mice reduced the accumulation of CD11b+Ly6ClowLy6G+ cells resembling granulocytic myeloid-derived suppressor cells (MDSCs) in both spleen and circulation. Additionally, IL-17 blockade reduced the recruitment of neutrophils into intestinal tissue, the expression and production of inflammatory cytokines, and the expression of intestinal STAT3. In addition, the anti-IL-17 treatment also reduced the expression of Arginase-1 and inducible nitric oxide synthase (iNOS) in the colon, both associated with the main suppressive activity of MDSCs. Thus, a lack of STAT1 signaling induces a significant change in the colonic microenvironment that supports inflammation and tumor formation. Anti-IL-17 treatment throughout the initial stages of CAC related to STAT1 deficiency abrogates the tumor formation possibly caused by myeloid cells.

Human interleukin-4-treated regulatory macrophages promote epithelial wound healing and reduce colitis in a mouse model.

Murine alternatively activated macrophages can exert anti-inflammatory effects. We sought to determine if IL-4-treated human macrophages [i.e., hM(IL4)] would promote epithelial wound repair and can serve as a cell transfer treatment for inflammatory bowel disease (IBD). Blood monocytes from healthy volunteers and patients with active and inactive IBD were converted to hM(IL4)s. IL-4 treatment of blood-derived macrophages from healthy volunteers and patients with inactive IBD resulted in a characteristic CD206+CCL18+CD14low/- phenotype (RNA-seq revealed IL-4 affected expression of 996 genes). Conditioned media from freshly generated or cryopreserved hM(IL4)s promoted epithelial wound healing in part by TGF, and reduced cytokine-driven loss of epithelial barrier function in vitro. Systemic delivery of hM(IL4) to dinitrobenzene sulphonic acid (DNBS)-treated Rag1-/- mice significantly reduced disease. These findings from in vitro and in vivo analyses provide proof-of-concept support for the development of autologous M(IL4) transfer as a cellular immunotherapy for IBD.

Use of STAT6 Phosphorylation Inhibitor and Trimethylglycine as New Adjuvant Therapies for 5-Fluorouracil in Colitis-Associated Tumorigenesis.

Colorectal cancer (CRC) is one of the most widespread and deadly types of neoplasia around the world, where the inflammatory microenvironment has critical importance in the process of tumor growth, metastasis, and drug resistance. Despite its limited effectiveness, 5-fluorouracil (5-FU) is the main drug utilized for CRC treatment. The combination of 5-FU with other agents modestly increases its effectiveness in patients. Here, we evaluated the anti-inflammatory Trimethylglycine and the Signal transducer and activator of transcription (STAT6) inhibitor AS1517499, as possible adjuvants to 5-FU in already established cancers, using a model of colitis-associated colon cancer (CAC). We found that these adjuvant therapies induced a remarkable reduction of tumor growth when administrated together with 5-FU, correlating with a reduction in STAT6-phosphorylation. This reduction upgraded the effect of 5-FU by increasing both levels of apoptosis and markers of cell adhesion such as E-cadherin, whereas decreased epithelial-mesenchymal transition markers were associated with aggressive phenotypes and drug resistance, such as ß-catenin nuclear translocation and Zinc finger protein SNAI1 (SNAI1). Additionally, Il-10, Tgf-ß, and Il-17a, critical pro-tumorigenic cytokines, were downmodulated in the colon by these adjuvant therapies. In vitro assays on human colon cancer cells showed that Trimethylglycine also reduced STAT6-phosphorylation. Our study is relatively unique in focusing on the effects of the combined administration of AS1517499 and Trimethylglycine together with 5-FU on already established CAC which synergizes to markedly reduce the colon tumor load. Together, these data point to STAT6 as a valuable target for adjuvant therapy in colon cancer.

Helminth-derived molecules inhibit colitis-associated colon cancer development through NF-κB and STAT3 regulation.

Inflammation is currently considered a hallmark of cancer and plays a decisive role in different stages of tumorigenesis, including initiation, promotion, progression, metastasis and resistance to antitumor therapies. Colorectal cancer is a disease widely associated with local chronic inflammation. Additionally, extrinsic factors such as infection may beneficially or detrimentally alter cancer progression. Several reports have noted the ability of various parasitic infections to modulate cancer development, favoring tumor progression in many cases and inhibiting tumorigenesis in others. The aim of our study was to determine the effects of excreted/secreted products of the helminth Taenia crassiceps (TcES) as a treatment in a murine model of colitis-associated colon cancer (CAC). Here, we found that after inducing CAC, treatment with TcES was able to reduce inflammatory cytokines such as IL-1ß, TNF-α, IL-33 and IL-17 and significantly attenuate colon tumorigenesis. This effect was associated with the inhibition of signal transducer and activator of transcription 3 and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation. Furthermore, we determined that TcES interfered with LPS-induced NF-κB p65 activation in human colonic epithelial cell lines in a Raf-1 proto-oncogene-dependent manner. Moreover, in three-dimensional cultures, TcES promoted reorganization of the actin cytoskeleton, altering cell morphology and forming colonospheres, features associated with a low grade of aggressiveness. Our study demonstrates a remarkable effect of helminth-derived molecules on suppressing ongoing colorectal cancer by downregulating proinflammatory and protumorigenic signaling pathways.

Taenia crassiceps-Excreted/Secreted Products Induce a Defined MicroRNA Profile that Modulates Inflammatory Properties of Macrophages.

Helminth parasites modulate immune responses in their host to prevent their elimination and to establish chronic infections. Our previous studies indicate that Taenia crassiceps-excreted/secreted antigens (TcES) downregulate inflammatory responses in rodent models of autoimmune diseases, by promoting the generation of alternatively activated-like macrophages (M2) in vivo. However, the molecular mechanisms triggered by TcES that modulate macrophage polarization and inflammatory response remain unclear. Here, we found that, while TcES reduced the production of inflammatory cytokines (IL-6, IL-12, and TNFα), they increased the release of IL-10 in LPS-induced bone marrow-derived macrophages (BMDM). However, TcES alone or in combination with LPS or IL-4 failed to increase the production of the canonical M1 or M2 markers in BMDM. To further define the anti-inflammatory effect of TcES in the response of LPS-stimulated macrophages, we performed transcriptomic array analyses of mRNA and microRNA to evaluate their levels. Although the addition of TcES to LPS-stimulated BMDM induced modest changes in the inflammatory mRNA profile, it induced the production of mRNAs associated with the activation of different receptors, phagocytosis, and M2-like phenotype. Moreover, we found that TcES induced upregulation of specific microRNAs, including miR-125a-5p, miR-762, and miR-484, which are predicted to target canonical inflammatory molecules and pathways in LPS-induced BMDM. These results suggest that TcES can modulate proinflammatory responses in macrophages by inducing regulatory posttranscriptional mechanisms and hence reduce detrimental outcomes in hosts running with inflammatory diseases.

Deficiency in STAT1 Signaling Predisposes Gut Inflammation and Prompts Colorectal Cancer Development.

Signal transducer and activator of transcription 1 (STAT1) is part of the Janus kinase (JAK/STAT) signaling pathway that controls critical events in intestinal immune function related to innate and adaptive immunity. Recent studies have implicated STAT1 in tumor⁻stroma interactions, and its expression and activity are perturbed during colon cancer. However, the role of STAT1 during the initiation of inflammation-associated cancer is not clearly understood. To determine the role of STAT1 in colitis-associated colorectal cancer (CAC), we analyzed the tumor development and kinetics of cell recruitment in wild-type WT or STAT1-/- mice treated with azoxymethane (AOM) and dextran sodium sulfate (DSS). Following CAC induction, STAT1-/- mice displayed an accelerated appearance of inflammation and tumor formation, and increased damage and scores on the disease activity index (DAI) as early as 20 days after AOM-DSS exposure compared to their WT counterparts. STAT1-/- mice showed elevated colonic epithelial cell proliferation in early stages of injury-induced tumor formation and decreased apoptosis in advanced tumors with over-expression of the anti-apoptotic protein Bcl2 at the colon. STAT1-/- mice showed increased accumulation of Ly6G⁺Ly6C-CD11b⁺ cells in the spleen at 20 days of CAC development with concomitant increases in the production of IL-17A, IL-17F, and IL-22 cytokines compared to WT mice. Our findings suggest that STAT1 plays a role as a tumor suppressor molecule in inflammation-associated carcinogenesis, particularly during the very early stages of CAC initiation, modulating immune responses as well as controlling mechanisms such as apoptosis and cell proliferation.

Parasites as negative regulators of cancer.

Several environmental factors (chemical, physical, and biological) can cause the initiation, promotion, and progression of cancer. Regarding the biological factors, several studies have found that infections caused by some bacteria, viruses and protozoan, and helminth parasites are related to carcinogenesis. However, in recent years a different approach has been implemented on the antitumor impact of parasitic diseases caused by some protozoan and helminths, mainly because such infections may affect several hallmarks of cancer, but the involved mechanisms still remain unknown. The beneficial effects reported for some parasitic diseases on tumorigenesis range from the induction of apoptosis, activation of the immune response, avoiding metastasis and angiogenesis, inhibition of proliferative signals, to the regulation of inflammatory responses that promote cancer. In this work, we reviewed the available information regarding how parasitic infections may modulate cancer progression. Despite the fact that specific mechanisms of action on tumors are not yet totally clear, we consider that detailed studies of the antitumor action of these organisms and their products could lead to the discovery and use of new molecules from these biological agents that may work as adjuvant therapy in the treatment of various types of cancer.

Extraintestinal Helminth Infection Limits Pathology and Proinflammatory Cytokine Expression during DSS-Induced Ulcerative Colitis: A Role for Alternatively Activated Macrophages and Prostaglandins.

Chronic inflammation of the intestinal mucosa is characteristic of inflammatory bowel diseases such as ulcerative colitis and Crohn's disease. Helminth parasites have developed immunomodulatory strategies that may impact the outcome of several inflammatory diseases. Therefore, we investigated whether Taenia crassiceps infection is able to decrease the inflammatory effects of dextran sulfate sodium- (DSS-) induced ulcerative colitis in BALB/c and C57BL/6 mice. Preinfection significantly reduced the manifestations of DSS-induced colitis, as weight loss and shortened colon length, and decreased the disease activity index independently of the genetic background of the mice. Taenia infection decreased systemic levels of proinflammatory cytokines while increasing levels of IL-4 and IL-10, and the inflammatory infiltrate into the colon was also markedly reduced. RT-PCR assays from colon showed that T. crassiceps-infected mice displayed increased expression of Arginase-1 but decreased expression of iNOS compared to DSS-treated uninfected mice. The percentages of T regulatory cells were not increased. The adoptive transfer of alternatively activated macrophages (AAMФs) from infected mice into mice with DSS-induced colitis reduced the severity of colon inflammation. Administration of indomethacin abrogated the anticolitic effect of Taenia. Thus, T. crassiceps infection limits the pathology of ulcerative colitis by suppressing inflammatory responses mechanistically associated with AAMФs and prostaglandins.

Extraintestinal helminth infection reduces the development of colitis-associated tumorigenesis.

Colitis-associated colorectal cancer (CAC) is one of the most common cancers and is closely related to chronic or deregulated inflammation. Helminthic infections can modulate inflammatory responses in some diseases, but their immunomodulatory role during cancer development remains completely unknown. We have analyzed the role of Taenia crassiceps-induced anti-inflammatory response in determining the outcome of CAC. We show that extraintestinal T. crassiceps infection in CAC mice inhibited colonic inflammatory responses and tumor formation and prevented goblet cell loss. There was also increased expression of IL-4 and alternatively activated macrophages markers in colonic tissue and negative immunomodulation of pro-inflammatory cytokine expression. In addition, T. crassiceps infection prevented the upregulation of ß-catenin and CXCR2 expression observed in the CAC mice, which are both markers associated with CAC-tumorigenesis, and reduced the numbers of circulating and colonic CD11b(+)Ly6C(hi)CCR2(+) monocytes. Thus, immunomodulatory activities induced by helminth infections may have a role in the progression of CAC.