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Aberrant lipid metabolism has recently been recognized as a new hallmark of malignancy, but the characteristics of fatty acid metabolism in breast cancer stem cells (BCSC) and potential interventions targeting this pathway remain to be addressed. Here, by using the in vitro BCSC models, mammosphere-derived MCF-7 cells and HMLE-Twist-ER cells, we found that the cells with stem cell-like properties exhibited a very distinct profile of fatty acid metabolism compared with that of their parental cancer cells, characterized by increased lipogenesis, especially the activity of stearoyl-CoA desaturase 1 (SCD1) responsible for the production of monounsaturated fatty acids, and augmented synthesis and utilization of the omega-6 arachidonic acid (AA). Suppression of SCD1 activity by either enzyme inhibitors or small interfering RNA (siRNA) knockdown strikingly limited self-renewal and growth of the BCSC, suggesting a key role for SCD1 in BCSC proliferation. Furthermore, elevated levels of SCD1 and other lipogenic enzymes were observed in human breast cancer tissues relative to the noncancer tissues from the same patients and correlated with the pathological grades. Interestingly, treatment of BCSC with omega-3 fatty acids, eicosapentaenoic acid and docosahexaenoic acid, effectively downregulated the expression of the lipogenic enzymes and markedly suppressed BCSC self-renewal and growth. Dietary supplementation of nude mice bearing BCSC-derived tumors with omega-3 fatty acids also significantly reduced their tumor load. These findings have demonstrated that increased lipogenesis is critical for self-renewal and growth of BCSC, and that omega-3 fatty acids are effective in targeting this pathway to exert their anticancer effect.

Background & Aims Epidemiological and animal studies have indicated an inverse correlation between the rising prevalence of obesity and metabolic syndrome and exposure to helminths. Whether helminth-induced immune response contributes to microbiota remodeling in obesity remains unknown. The aim of this study is to explore the immune-regulatory role of helminth in the prevention of HFD-induced obesity through remodeling gut microbiome. Methods C57BL/6J WT and STAT6-/- mice were infected with Heligmosomoides polygyrus and followed by high fat diet (HFD) feeding for 6 weeks. The host immune response, body weight, and fecal microbiota composition were analyzed. We used adoptive transfer of M2 macrophages and microbiota transplantation approaches to determine the impact of these factors on HFD-obesity. We also examined stool microbiota composition and short chain fatty acids (SCFAs) concentration and determined the expression of SCFA-relevant receptors in the recipient mice. Results Helminth infection of STAT6-/- (Th2-deficient) mice and adoptive transfer of helminth-induced alternatively activated (M2) macrophages demonstrated that the helminth-associated Th2 immune response plays an important role in the protection against obesity and induces changes in microbiota composition. Microbiota transplantation showed that helminth-induced, Th2-dependent alterations of the gut microbiota are sufficient to confer protection against obesity. Collectively, these results indicate that helminth infection protects against HFD-induced obesity by Th2-dependent, M2 macrophage-mediated alterations of the intestinal microbiota. Conclusion Our findings provide new mechanistic insights into the complex interplay between helminth infection, the immune system and the gut microbiota in a HFD-induced obesity model and holds promise for gut microbiome-targeted immunotherapy in obesity prevention., Graphical abstract

Scope:Sonchus Oleraceus, named bitter vegetable (BV), has been known to have multiple health benefits such as anti-aging and anti-inflammation. However, the role of BV in the prevention of obesity is unclear. The aim of this study was to examine the effect of BV lipid extracts (BVL) on obesity development.Methods and Results: Following treatments of high fat diet-induced obese mice (C57BL/6J) with BVL (0.3 mg/g of BW per mouse) for a month, mice exhibited a significant reduction in weight gain, blood triglyceride, and fasting blood glucose compared to control mice. Intriguingly, phosphorylated AMPK, a key regulator of nutrient metabolism, was markedly increased in inguinal fat of BVL group. In 3T3-L1 cells, BVL-7 (100 μg/ml), an omega-3 fatty acid-rich fraction from BVL, lowered lipid accumulation, and down-regulated the gene expression of adipocyte markers. The inhibitory effect of BVL occurred at the early stage of adipocyte differentiation, leading to the delay of mitotic clonal expansion. AMPK knockdown by siRNA abolished the inhibitory effect of BVL-7 on adipogenesis, suggesting that AMPK is essential for BVL-regulated adipocyte differentiation.Conclusion: BVL can effectively inhibit adipogenesis through, at least in part, stimulating AMPK pathway and attenuate HFD-induced obesity. Our findings suggest that BVL can be a promising dietary supplement for protection against obesity, and the effective component of BVL can be potentially developed as anti-obesity drugs.

Inflammatory bowel disease (IBD) increases the risk of colorectal cancer, and it has the potential to diminish the quality of life. Recent clinical and experimental evidence demonstrate protective aspects of parasitic helminth infection against IBD. Reports have highlighted the potential use of helminths and their byproducts as potential treatment for IBD. In the current study, we studied the effect of a newborn larvae-specific serine protease from Trichinella spiralis (TsSp) on the host immune and inflammatory responses. A 49-kDa recombinant TsSp (rTsSp) was expressed in Escherichia coli BL21 (DE3) and purified. The cytotoxicity of rTsSp was analyzed. The immune protective effect of rTsSp was studied by using dextran sodium sulfate (DSS)-induced mouse colitis model. The result illustrated that rTsSp has no toxic effects on cells. We further demonstrated that administration of the rTsSp without the additional adjuvant before the induction of DSS-induced colitis reduced the severity of intestinal inflammation and the disease index; it suppressed macrophage infiltration, reduced TNF-α secretion, and induced IL-10 expression. Our findings suggest therapeutic potential of rTsSp on colitis by altering the effect of macrophages. Data also suggest immunotherapy with rTsSp holds promise for use as an additional strategy to positively modulate inflammatory processes involved in IBD.

Hyaluronan is a glycosaminoglycan polymer that has been shown to play an important role in homeostasis of the gastrointestinal tract. However, its mechanistic significance in gastrointestinal epithelial barrier elements remain unexplored. Here, our results revealed that hyaluronan treatment resulted in significant changes in the gut microbiota in mice. To demonstrate the functional consequences of hyaluronan-treatment and hyaluronan-induced microbiota alterations, Citrobacter rodentium- and DSS-induced colitis models and microbiota transplantation approaches were utilized. We showed that hyaluronan alleviated intestinal inflammation in both pathogen and chemically induced intestinal mucosal damage. The protection in bacterial colitis was associated with enhanced C. rodentium clearance and alleviation of pathogen-induced gut dysbiosis. Microbiota transplantation experiments showed that the hyaluronan-altered microbiota is sufficient to confer protection against C. rodentium infection. Colonization with Akkermansia muciniphila, a commensal bacterium that is greatly enriched by hyaluronan treatment, alleviated C. rodentium-induced bacterial colitis in mice. Additionally, Akkermansia-induced protection was found to be associated with the induction of goblet cells and the production of mucins and epithelial antimicrobial peptides. Collectively, these results provide novel insights into the regulatory role of hyaluronan in modulating the gut microbiota and immunity in enteric infection and inflammation, with therapeutic potential for gut microbiome-targeted immunotherapy.

Infection with the intestinal helminth parasite Heligmosomoides polygyrus exacerbates the colitis caused by the bacterial enteropathogen Citrobacter rodentium. To clarify the underlying mechanism, we analyzed fecal microbiota composition of control and helminth-infected mice and evaluated the functional role of compositional differences by microbiota transplantation experiments. Our results showed that infection of Balb/c mice with H. polygyrus resulted in significant changes in the composition of the gut microbiota, characterized by a marked increase in the abundance of Bacteroidetes and decreases in Firmicutes and Lactobacillales. Recipients of the gut microbiota from helminth-infected wide-type, but not STAT 6-deficient, Balb/c donors had increased fecal pathogen shedding and significant worsening of Citrobacter-induced colitis compared to recipients of microbiota from control donors. Recipients of helminth-altered microbiota also displayed increased regulatory T cells and IL-10 expression. Depletion of CD4+CD25+ T cells and neutralization of IL-10 in recipients of helminth-altered microbiota led to reduced stool C. rodentium numbers and attenuated colitis. These results indicate that alteration of the gut microbiota is a significant contributor to the H. polygyrus-induced exacerbation of C. rodentium colitis. The helminth-induced alteration of the microbiota is Th2-dependent and acts by promoting regulatory T cells that suppress protective responses to bacterial enteropathogens.

Summary Signaling abnormalities in immune responses in the small intestine can trigger chronic type 2 inflammation involving interaction of multiple immune cell types. To systematically characterize this response, we analyzed 58,067 immune cells from the mouse small intestine by single-cell RNA sequencing (scRNA-seq) at steady state and after induction of a type 2 inflammatory reaction to ovalbumin (OVA). Computational analysis revealed broad shifts in both cell-type composition and cell programs in response to the inflammation, especially in group 2 innate lymphoid cells (ILC2s). Inflammation induced the expression of exon 5 of Calca, which encodes the alpha-calcitonin gene-related peptide (α-CGRP), in intestinal KLRG1+ ILC2s. α-CGRP antagonized KLRG1+ ILC2s proliferation but promoted IL-5 expression. Genetic perturbation of α-CGRP increased the proportion of intestinal KLRG1+ ILC2s. Our work highlights a model where α-CGRP-mediated neuronal signaling is critical for suppressing ILC2 expansion and maintaining homeostasis of the type 2 immune machinery.

Objectives Reducing the increased number of white adipocyte progenitors (WAP) is considered a novel approach to controlling obesity. The role of omega-3 polyunsaturated fatty acids (PUFA) in regulating the WAP resident population is unclear. The objective of this study was to investigate the effect of omega-3 PUFA on the niche composition of adipose-derived stem cells. Methods Stromal vascular cell fraction (SVF) was collected from subcutaneous fat of wild-type (WT) and transgenic mice carrying a fat-1 gene from Caenorhabditis elegans (Fat-1 mice), which are capable of synthesizing omega-3 PUFA and have much higher tissue levels of omega-3 PUFA relative to WT mice. The isolated SVF cells were cultured and used for the examination of adipocyte differentiation, adipogenic markers, fatty acid composition, and WAP numbers. Results SVF isolated from Fat-1 mice (Fat-1-SVF) exhibited markedly fewer differentiated adipocytes with smaller cell size and less lipid content than that of WT mice (WT-SVF). Accordingly, adipogenesis-related genes and the white adipocyte surface marker ASC-1 were downregulated in Fat-1-SVF relative to WT-SVF. Furthermore, WAP numbers and adipose tissue macrophages were lower in Fat-1-SVF than WT-SVF. Conclusions Omega-3 PUFA can both limit the WAP resident population and suppress their differentiation to white adipocytes, suggesting a new mechanism for the antiobesity effect of omega-3 PUFA.

Epidemiological studies indicate an inverse correlation between the prevalence of the so-called western diseases, such as obesity and metabolic syndrome, and the exposure to helminths. Obesity, a key risk factor for many chronic health problems, is rising globally and is accompanied by low-grade inflammation in adipose tissues. The precise mechanism by which helminths modulate metabolic syndrome and obesity is not fully understood. We infected high fat diet (HFD)-induced obese mice with the intestinal nematode parasite Heligmosomoides polygyrus and observed that helminth infection resulted in significantly attenuated obesity. Attenuated obesity corresponded with marked upregulation of uncoupling protein 1 (UCP1), a key protein involved in energy expenditure, in adipose tissue, suppression of glucose and triglyceride levels, and alteration in the expression of key genes involved in lipid metabolism. Moreover, the attenuated obesity in infected mice was associated with enhanced helminth-induced Th2/Treg responses and M2 macrophage polarization. Adoptive transfer of helminth-stimulated M2 cells to mice that were not infected with H. polygyrus resulted in a significant amelioration of HFD-induced obesity and increased adipose tissue browning. Thus, our results provide evidence that the helminth-dependent protection against obesity involves the induction of M2 macrophages.

BACKGROUND Intestinal inflammation is associated with systemic translocation of commensal antigens and the consequent activation of B and T lymphocytes. The long-term consequences of such immune activation are not completely understood. METHODS C57BL/6 mice were subjected to 2 courses of treatment with dextran sulfate sodium (DSS) to induce colitis. Two to 7 weeks after the DSS treatment, the mice were infected intraperitoneally with Salmonella enterica serovar Typhimurium. The outcome of infection was evaluated based on survival and tissue pathogen burden. RESULTS Mice that had recovered from DSS colitis displayed a significant increase in resistance to S. Typhimurium infection as indicated by improved survival and decreased tissue pathogen numbers. The colitis-induced increase in resistance to systemic salmonellosis lasted for as long as 7 weeks after discontinuing DSS and was dependent on T lymphocytes but not on B cells. Interestingly, depletion of CD4 and CD8 T cells just before the Salmonella infection did not alter the colitis-induced increase in resistance. Mice that had recovered from colitis had evidence of persistent activation of resident peritoneal macrophages and enhanced Salmonella-induced neutrophil recruitment to the peritoneum. Macrophage depletion with clodronate liposomes abrogated the colitis-induced increase in resistance to Salmonella. CONCLUSIONS Taken together, our results indicate that DSS colitis leads to a long-lasting increase in resistance to Salmonella infection that is initiated in a T cell-dependent manner but is ultimately mediated independently of B and T cells as a result of persistent changes in innate immune cell function.

Autophagy is an important mechanism used by macrophages to kill intracellular pathogens. The results reported in this study demonstrate that autophagy is also involved in the macrophage killing of the extracellular enteropathogen Citrobacter rodentium after phagocytosis. The process was significantly impaired in macrophages isolated from mice chronically infected with the helminth parasite Heligmosomoides polygyrus. The H. polygyrus-mediated inhibition of autophagy was Th2 dependent because it was not observed in macrophages isolated from helminth-infected STAT6-deficient mice. Moreover, autophagy of Citrobacter was inhibited by treating macrophages with IL-4 and IL-13. The effect of H. polygyrus on autophagy was associated with decreased expression and processing of L chain protein 3 (LC3), a key component of the autophagic machinery. The helminth-induced inhibition of LC3 expression and processing was STAT6 dependent and could be recapitulated by treatment of macrophages with IL-4 and IL-13. Knockdown of LC3 significantly inhibited autophagic killing of Citrobacter, attesting to the functional importance of the H. polygyrus-mediated downregulation of this process. These observations reveal a new aspect of the immunosuppressive effects of helminth infection and provide mechanistic insights into our earlier finding that H. polygyrus significantly worsens the in vivo course of Citrobacter infection.

Chronic infection with intestinal helminth parasites is a major public health problem, particularly in the developing world, and can have significant effects on host physiology and the immune response to other enteric infections and antigens. The mechanisms underlying these effects are not well understood. In the current study, we investigated the impact of infection with the murine nematode parasite Heligmosomoides polygyrus , which resides in the duodenum, on epithelial barrier function in the colon. We found that H. polygyrus infection produced a significant increase in colonic epithelial permeability, as evidenced by detection of elevated serum levels of the tracer horseradish peroxidase following rectal administration. This loss of normal barrier function was associated with clear ultrastructural changes in the tight junctions of colonic epithelial cells and an alteration in the expression and distribution of the junctional protein E-cadherin. These parasite-induced abnormalities were not observed in SCID mice but did occur in SCID mice that were adoptively transferred with wild-type T cells, indicating a requirement for adaptive immunity. Furthermore, the helminth-induced increase in gut permeability was not seen in STAT6 knockout (KO) mice. Taken together, the results demonstrate that one of the mechanisms by which helminths exert their effects involves the lymphocyte- and STAT6-dependent breakdown of the intestinal epithelial barrier. This increase in epithelial permeability may facilitate the movement of lumenal contents across the mucosa, thus helping to explain how helminth infection can alter the immune response to enteric antigens.

In the small intestine, a niche of accessory cell types supports the generation of mature epithelial cell types from intestinal stem cells (ISCs). It is unclear, however, if and how immune cells in the niche affect ISC fate or the balance between self-renewal and differentiation. Here, we use single-cell RNA sequencing (scRNA-seq) to identify MHC class II (MHCII) machinery enrichment in two subsets of Lgr5⁺ ISCs. We show that MHCII⁺ Lgr5⁺ ISCs are non-conventional antigen-presenting cells in co-cultures with CD4⁺ T helper (Th) cells. Stimulation of intestinal organoids with key Th cytokines affects Lgr5⁺ ISC renewal and differentiation in opposing ways: pro-inflammatory signals promote differentiation, while regulatory cells and cytokines reduce it. In vivo genetic perturbation of Th cells or MHCII expression on Lgr5⁺ ISCs impacts epithelial cell differentiation and IEC fate during infection. These interactions between Th cells and Lgr5⁺ ISCs, thus, orchestrate tissue-wide responses to external signals. Intestinal stem cells act as non-conventional antigen presenting cells, and these interactions with T helper cells modulate ISC renewal and differentiation to shape the intestine. Keywords: gut biology; intestinal stem cells; ISCs; T helper; Th; mucosal immunity; MHC class II; MHCII; tuft cells; T regulatory; Treg; single cell RNA-seq; scRNA-seq; epithelial differentiation; stem cell renewal, National Institute of Health (U.S.) (Award 1DP2OD020839), National Institute of Health (U.S.) (Grant CA211184), National Institute of Health (U.S.) (Grant AG045144)

Infections with intestinal helminth and bacterial pathogens, such as enteropathogenic Escherichia coli, continue to be a major global health threat for children. To determine whether and how an intestinal helminth parasite, Heligomosomoides polygyrus, might impact the TLR signaling pathway during the response to a bacterial enteropathogen, MyD88 knockout and wild-type C57BL/6 mice were infected with H. polygyrus, the bacterial enteropathogen Citrobacter rodentium, or both. We found that MyD88 knockout mice co-infected with H. polygyrus and C. rodentium developed more severe intestinal inflammation and elevated mortality compared to the wild-type mice. The enhanced susceptibility to C. rodentium, intestinal injury and mortality of the co-infected MyD88 knockout mice were found to be associated with markedly reduced intestinal phagocyte recruitment, decreased expression of the chemoattractant KC, and a significant increase in bacterial translocation. Moreover, the increase in bacterial infection and disease severity were found to be correlated with a significant downregulation of antimicrobial peptide expression in the intestinal tissue in co-infected MyD88 knockout mice. Our results suggest that the MyD88 signaling pathway plays a critical role for host defense and survival during helminth and enteric bacterial co-infection., Author Summary Infections with intestinal helminths and enteric bacterial pathogens such as enteropathogenic Escherichia coli (EPEC) continue to be major global health problems, especially for children. The ability of the host to control bacterial enteropathogens may be influenced by host immune status and by concurrent infections. Helminth parasites are of particular interest in this context because of their ability to modulate host immune responses, and because their geographic distribution coincides with those parts of the world where infections caused by bacterial enteropathogens are most problematic. In this study, we determined how intestinal helminth infection regulates host innate immunity against bacterial enteropathogens by using a murine co-infection model. This model involves co-infection with the intestinal nematode parasite Heligmosomoides polygyrus and the Gram-negative bacterial pathogen Citrobacter rodentium, the murine equivalent of EPEC. The infections were carried out in wild-type mice and in mice lacking MyD88, a protein required for signaling by the Toll-like receptors. We found that co-infection with the helminth parasite significantly worsened Citrobacter-induced colitis in the MyD88-deficient mice, in association with increased mortality and compromised innate immune responses. Our observations demonstrate an important role for MyD88-dependent and -independent signaling in host survival during helminth and enteric bacterial co-infection.

Background and Aim:Murine norovirus (MNV) infection was shown to induce histopathological changes in the intestine of conventional housed mice or lethal systemic infection in immunocompromised mice. The mechanism which triggered the inflammation is not well understood. In the present study, the influence of MNV infection on histological and immunological characteristics of mucosal inflammation in germfree and Schaedler flora colonized IL10-deficient (Il10-/-) mouse model was examined. Methods: Germfree (GF) C57Bl/6J-Il10-/mice and GF mice colonized with Schaedler Flora were monitored after MNV infection for structural and functional intestinal barrier changes using histology, RTqPCR, ELISA and TUNEL assay. Results: No inflammatory lesions were observed in GF B6Il10-/and MNV-infected mice whereas in MNV-infected GF mice colonized with Schaedler Flora inflammatory lesions were detected comparable to SPF mice infected with MNV. Interferon inducible genes were increased in the small intestine of GFmice 48h after infection. However, four weeks after infection GF mice showed no increased IFNgamma production, whereas in Schaedler Flora colonized mice IFNgamma production was increased similar to SPF infected mice. Although there is no reduction of gene expression of tight junction molecules and an enhanced rate of epithelial apoptosis 48h after infection of GF mice the expression level changed within 4 weeks in MNV infected Schaedler Flora. Conclusions: MNV induces inflammation and epithelial barrier disruption that depends on the presence of the bacteria. Thus, MNV and enteric microbiota together represent a potent colitogenic stimulus and inflammatory trigger.