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Sterile neutrophilia (SN) is commonly associated with chronic disease, but its role in inflammatory bowel diseases (IBD) is largely unknown. AMD3100 (plerixafor; AMD) is a CXCR4 antagonist that promotes neutrophil egress from bone marrow resulting in SN. To study the role of SN in IBD, we induced chronic intestinal inflammation in C57BL6 mice via 4 weekly injections of IL-10R neutralizing antibody and administered AMD (10 mg/kg bw, i.p., twice weekly) or vehicle. As expected, AMD-treated control and colitic mice had increased peripheral and splenic neutrophils. AMD treatment remarkably reduced epithelial damage and hyperplasia, immune cell infiltration, and disease activity index. To elucidate the protective mechanism, we analyzed immune cells in peripheral circulation, spleen and lamina propria. In circulation, colitic mice with AMD treatment had elevated % CD3+CD4+ helper T cells (Th) but reduced % CD3+CD8+ cytotoxic T (Tc) cells. Yet, the % CD3+CD4+FoxP3+ cells (Tregs) and % CD3+CD4+RORγt+ cells (Th17) remained unchanged. In spleen, AMD treatment significantly increased Tregs, but decreased Th and Tc cells. In lamina propria, AMD treatment markedly elevated Tregs, including RORγt+ Tregs and tolerogenic IL-10+ Tregs. Conversely, the inflammatory IL-17+ Th cells were decreased in AMD-treated colitic mice. In addition to the immunosuppressive milieu, AMD-treated colitic mice had increased ileal expression of the antimicrobial peptide angiogenin 4, which further suggests SN to be important in maintaining gut microbiota homeostasis. Collectively, our results demonstrate that SN could be an adaptive and beneficial immune response during chronic intestinal inflammation. This research was funded by R01 grant from the National Institutes of Health (NIH) [grant number CA219144] and Crohn’s and Colitis Foundation (CCF) [grant number 855256]. more...

Breast milk is the preferred food for the newborns and considered as complete ‘edible immune system’. In addition, breast milk is a source of innate and adaptive proteins which not only protects the naïve infant gastrointestinal tract from enteropathogens but also aid in the appropriate initial colonization of gut microbiota. In this study, we analyzed antimicrobial immune proteins in the milk from C57BL/6 dams at several time points, and observed distinctive patterns in the immune protein levels throughout the lactation period. The presence of innate immune proteins serum amyloid A (SAA), CD14, and notably lipocalin-2 (Lcn2) were observed in high quantities with Lcn2 and SAA present at microgram levels suggesting a potential niche for these proteins during neonatal immune development. Moreover, low dose LPS administration to dams significantly increased some of these proteins in the milk. In addition, adaptive immune proteins immunoglobulins (Ig) A and G with IgG were present in milk at higher quantities compared to IgA at day 5 post-delivery. Interestingly, class-switching occurs by day 15 indicating a selective adaptive immune preference towards IgA, which could be due to gut colonization of newborns with more complex gut microbiota with aging and IgA being a major gut homeostatic factor. Additionally, milk from IgA-deficient dams allowed for robust proliferation of E. coli compared to wild-type milk further signifying the potential involvement of IgA during the early colonization of microbes in the neonatal gut. Collectively, our findings provide insight in the various immune proteins with antimicrobial activity may play a major role in the initial microbial colonization in the gut, which has long-lasting consequences on the host. Supported by NIH R01-CA219144, NCI Diversity Supplement (CA219144-05S1) more...

The inflammasome cytokine IL-1b is an important mediator of the inflammatory responses against invading pathogens. However, it is not clear whether sustained IL-1b signaling following the loss of its endogenous inhibitor, secretory IL-1 receptor antagonist (sIL-1Ra), could improve mucosal immunity against the enteropathogen, Citrobacter rodentium (CR). In light of increased prescription of rec-sIL-1Ra (aka Anakinra) to treat inflammatory bowel disease, we undertook this study. At basal levels, sIL-1Ra-deficient (sIL-1RaKO) mice displayed leukocytosis and elevated inflammatory marker, i.e. serum and fecal lipocalin 2. Furthermore, bone marrow-derived macrophages and neutrophils from sIL-1RaKO mice generated higher levels of iNOS and nitrite, and ROS and NETs, respectively. These results collectively suggest sIL-1RaKO mice have low-grade chronic inflammation. Based on these results, we hypothesized that sIL-1RaKO mice with persistent IL1-b signaling could efficiently clear CR infection than their WT littermates. Oral challenge of CR (1×109 CFU/mouse) resulted in luminal colonization, which peaked at day 7 post-infection, in both groups; however, sIL-1RaKO mice displayed a higher CR burden and an exacerbated infection. The aggravated course of infection was further visualized by inoculating sIL-1RaKO mice with bioluminescent CR. Histologic analysis revealed that transmissible colonic hyperplasia was more pronounced in sIL-1RaKO mice. Interestingly, basal ileal Paneth’s cell-specific antimicrobial proteins (Ang4 and Reg3g) were significantly reduced in sIL-1RaKO mice. Collectively, our results demonstrate that a balanced mucosal IL-1b signaling is required to counter and clear enteropathogen infection. more...

Inflammatory bowel diseases (IBD) consisting of Crohn’s and ulcerative colitis are resultant of dysregulation of the immune system leading to intestinal inflammation and microbial dysbiosis. Numerous studies in recent years highlighted the pivotal role of gut microbiota and their metabolites in host physiological processes including in IBD. Urolithin A (UroA) is a microbial metabolite derived from polyphenolics (e.g., ellagitannins/ellagic acid) of pomegranate and berries. We also synthesized a potent structural analogue of UroA (UAS03) and tested their efficacies in preventing and treating colitis in pre-clinical models. Our studies showed that UroA/UAS03 significantly enhance gut barrier function in addition to blocking unwarranted inflammation. We demonstrate that UroA and UAS03 exert their barrier functions through activation of aryl hydrocarbon receptor (AhR)- nuclear factor erythroid 2–related factor 2 (Nrf2)-dependent pathways to upregulate epithelial tight junction proteins. In addition, treatment with these compounds attenuated colitis in pre-clinical models by remedying barrier dysfunction and blocking increased inflammatory mediators such as IL-6, TNF-α and IL-1β. UroA/UAS03 failed to induce tight junction proteins and protect against 2,4,6-Trinitrobenzenesulfonic acid (TNBS)-induced colitis in AhR−/− and Nrf2−/− mice suggesting an obligatory requirement of AhR and Nrf2 pathways for UroA/UAS03 mediated beneficial activities. Overall, the results highlight how microbial metabolites provide two-pronged beneficial activities at gut epithelium by enhancing barrier functions and reducing systemic and local inflammation to protect from colonic diseases. more...

Dietary fermentable, soluble fibers (e.g. inulin) are known for promoting health; yet, ~40% of Toll-like receptor 5 deficient (T5KO) mice fed inulin-containing diet (ICD) develop icteric hepatocellular carcinoma (HCC) (Cell, 2018). Specifically, the disease progresses starting with hepatic cholestasis and neutrophilic inflammation at one month, followed by HCC at 6 months of ICD feeding. This diet-driven HCC was dependent on the gut microbiota and was mediated, largely in part, via surfeit production of immunosuppressive short-chain fatty acids (SCFA) and secondary bile acids, presumably as compensatory mechanisms against inflammation in the 40% pre-HCC T5KO mice. What was most striking was the upregulation of systemic bilirubin (a host immunosuppressive metabolite) within one week of ICD feeding. The hyperbilirubinemia was accompanied with an increase in IgA by ~50 and ~10-fold in the serum and liver, respectively, compared to the 60% non-HCC T5KO mice. Markers for anti-tumor T cell exhaustion, i.e. hepatic PD-L1 and layilin, were also upregulated, where the former was elevated within a week of feeding and persisted, while the latter was observed at 6 months. Hepatic immune cell phenotyping after the onset of HCC revealed substantial increase in CD4+FOXP3+PD-L1+T cells and IgA+IL10+PD-L1+Bcells, with the latter denoting a recently identified subset of highly immunosuppressive B cells. Our unprecedented findings collectively demonstrate that dietary fibers and SCFA —which are widely-held to be anti-inflammatory— could potentiate immunosuppression and impede anti-tumor immune surveillance in the tumor microenvironment in mice with gut microbiota dysbiosis. more...

Iron is a proinflammatory transition metal ion, yet limited information is documented on the differences in iron homeostasis under Th1 and Th2 biased conditions. We used C57BL/6 (BL6) and Balb/C (BB) strains, which exhibit prototypical Th1 and Th2 immune responses, to investigate the extent to which Th1 and Th2 biased responses dictate iron homeostasis during healthy and inflammatory conditions. Systemic and tissue iron levels were assessed in age and gender matched BL6 and BB mice. Gene expression for iron absorption, transport, and storage proteins were quantified in duodenum and liver. Intracellular labile iron pool (LIP) was assessed in bone marrow-derived macrophages (BMDM) and neutrophils (BMDN). Hypoferremia of inflammation was assessed by measuring serum iron before and after LPS treatment. BB mice displayed significantly higher levels of systemic and storage iron than BL6 mice. Although the expression of hepcidin and ferroportin were comparable between the two strains, hepcidin-independent iron absorption is more prominent in BB mice, as indicated by higher expression of genes involved in iron absorption and transport: DcytB, DMT1, SFT, and hephaestin. Similarly, hepatic TfR1, TfR2, transferrin, and DMT1 expression was higher in BB mice. Moreover, the basal levels of LIP in BB BMDM and BMDN were 3.4 and 2.6 folds higher than BL6, respectively. The LPS-induced hypoferremia of inflammation is more pronounced in BL6 than BB mice. Taken together, iron homeostasis is distinct between these two strains during both healthy and inflammatory conditions. BB mice showed more iron load than BL6 mice, which could be explained, in part, by the higher expression of iron uptake genes and also reduced Th1 responses in BB mice. more...

Iron, an essential transition metal ion, is required to facilitate the antimicrobial and redox activity of heme proteins expressed in macrophages (Mφs). In this study, we investigated whether the chelation of iron by enterobactin (Ent; a prototypical bacterial siderophore) can affect the immune responses of macrophages. Herein, we demonstrated that exogenous Ent confers protection to the intracellular pathogen Salmonella enterica serovar Typhimurium from Mφs by promoting M1 to M2 polarization, reducing the expression of pro-inflammatory cytokines and impairing their intracellular killing. Accordingly, WT Salmonella survived better compared to Ent-deficient strain (ΔentB) in Mφs. The addition of apo-Ent (iron-free), but not ferric-Ent, rescued both strains, thus suggesting that the immuno-modulatory effect of Ent could be likely mediated via chelation of Mφs cellular iron. To determine whether the effects of Ent on Mφs could be due to altered cell viability, we next assessed the Mφs for markers of apoptosis via flow cytometry. Interestingly, we observed that apo-Ent, but not ferric-Ent, promoted a time-dependent apoptosis in Mφs, which can be detected as early as 12h after Ent treatment. These findings corroborated with the time-dependent release of lactate dehydrogenase (cytotoxicity marker) from Ent-treated Mφs. Collectively, our findings demonstrate that Ent not only facilitates bacterial iron uptake but also serves to safeguard bacteria against Mφ innate immune responses, which could be mediated, in part, by inducing delayed cellular apoptosis. more...

Neutrophil extracellular traps (NETs), DNA structures released from neutrophils, are primarily enriched with bactericidal proteins. We envision that the antimicrobial nature of NETs would be an appropriate immune response in the gut, which is continuously exposed to trillions of bacteria. NETs formation is peptidyl arginine deiminase-4 (PAD4)-dependent, therefore, we employed Pad4KO mice and their WT littermates in two well-established models of murine colitis, i.e., loss of IL-10 signaling (immune hyperactivation) and chemical-induced injury (DSS). Upon treatment with αIL-10R mAb, Pad4KO mice developed robust chronic colitis as exemplified by colomegaly, colonic crypt elongation, and increased immune cell infiltration. In comparison, αIL-10R-treated WT mice exhibited modest colitis. We theorized that NETs may function as a ‘safety net’ which confine the pro-inflammatory neutrophil granule proteins (NGP) and prevent them to cause further damage. Indeed, the concentration and activity of NGP [i.e. lipocalin 2 (Lcn2), myeloperoxidase (MPO) and neutrophil elastase (NE)], in serum and in colons, were strikingly elevated in colitic Pad4KO mice, when compared to WT mice. The level of colonic secretory leukocyte protease inhibitor (SLPI) was reduced in the colitic Pad4KO mice. Such impaired SLPI response coupled with the ‘spillover’ of NGPs may, in part, explain the aggravated colitis in mice lacking NETs. Similarly, upon DSS treatment Pad4KO mice developed more severe chronic colitis than WT mice. Taken together, our findings advance the notion that gastrointestinal NETs are beneficial, and elucidation of NETs-mediated signaling, during colitis, may yield a novel therapeutic approach to mitigate intestinal inflammation. more...

Background Iron is highly reactive and could induce oxidative stress via augmenting the activity of heme enzymes, i.e., NADPH oxidase and myeloperoxidase (MPO). Unlike the membrane-restricted NADPH oxidase, MPO remains active upon its release into circulation and thus may potentially exacerbate iron-induced oxidative damage. Objective To investigate the extent to which MPO is involved in iron-induced toxicity. Methods Acute toxicity was induced by intraperitoneal injection of FeSO4 to MPO deficient (MPOKO) mice and their WT littermates. In some studies, WT mice were pretreated with 4-aminobenzoic acid hydrazide (a MPO inhibitor). Chronic toxicity was induced by feeding 2% carbonyl iron-diet to WT and MPOKO mice. Iron status, MPO levels, inflammatory (SAA, lipocalin 2, KC, IL-6 and IL-1β), organ damage (AST, ALT) and oxidative stress (MDA) markers were assessed. Results Acute iron administration upregulated circulating MPO in WT mice, which may be due to increased neutrophil degranulation or oxidative burst. However, the loss of MPO activity in mice, either via inhibition or genetic deficiency, markedly reduced iron-induced organ damage and systemic inflammation. In contrast, long-term iron feeding to WT mice reduced circulating MPO, but promoted its accumulation in liver. While both MPOKO and WT mice showed similar levels of diet-induced hyperferremia, MPOKO mice displayed significantly reduced inflammatory response and oxidative stress. Conclusion Genetic deficiency or pharmacologic inhibition of MPO substantially attenuated acute and chronic iron-induced toxicity. Our results suggest that targeting MPO during iron overload may be a promising approach to reduce iron-induced toxicity/side effects. more...

Peptidyl arginine deiminase-4 (PAD4) is required for the formation of neutrophil extracellular traps (NETs), which is part of the host antimicrobial innate immune response. However, the role of NETs in the pathogenesis and clearance of Citrobacter rodentium (a well-characterized mouse enteropathogen) infection remains unclear. In this study, we challenged the PAD4-deficient (Pad4KO; NETs-deficient) and WT littermates with C. rodentium. Luminal colonization of C. rodentium in Pad4KO mice peaked between 11-14 days post-infection (pi), whereas WT mice cleared the infection with no detectable level of the pathogen in fecal shedding after 7-10 days. Bacterial dissemination to spleen and mesenteric lymph node was significantly higher in Pad4KO mice at day 8 and 28 pi compared to WT. Additionally; Pad4KO mice displayed splenomegaly, colomegaly and elevated colonic and systemic pro-inflammatory markers (lipocalin 2 and serum amyloid A). Histological analysis demonstrated that transmissible colonic hyperplasia and goblet cell depletion were more pronounced in Pad4KO mice. Pad4KO mice also exhibited epithelial barrier dysfunction as indicated by their heightened serum immunoreactivity to bacterial proteins. Flow cytometric analysis revealed a substantial reduction in IL-10-producing macrophages and Th17 cells in the lamina propria and spleens of Pad4KO mice compared to WT mice. However, colonic expression of IL-17A and IL-22 were highly elevated in Pad4KO mice on day 8 pi, but were attenuated on day 28 both in Pad4KO and WT mice. Taken together, our findings highlight that NETs are indispensable in promoting clearance of C. rodentium and resolving the associated intestinal inflammation. more...

The host immune system is constantly exposed to diverse microbial ligands, including flagellin (FliC; a ligand for TLR5 and NLRC4) and lipopolysaccharide (LPS; a ligand for TLR4), whose ability to induce immune tolerance to subsequent exposure is well-documented in vitro. In this study, we investigated the extent to which FliC and LPS could induce immune cross-tolerance to one another in vivo, as well as the interaction between the innate and adaptive immune response to FliC. Mice pre-treated with LPS displayed attenuated serum keratinocyte-derived chemokine [KC; alias interleukin (IL)-8] and IL-6 response to FliC; however, the administration of FliC followed by LPS instead resulted in an additive response. LPS also augmented the serum IL-18 response to FliC; the increment was more pronounced when LPS was administered before, than after, FliC. Intriguingly, the generation of anti-FliC antibodies was notably impaired in mice sequentially challenged with LPS and FliC, compared to mice given only FliC. A strong negative correlation between high anti-FliC titer and reduced KC, IL-6 and IL-18 responses to FliC re-challenge was observed in these mice, suggesting that adaptive immunity could tolerize TLR5 and NLRC4. Indeed, the serum from WT mice with high anti-FliC titer, but not Rag1KO mice, was able to dampen FliC-induced secretion of IL-8 from the human epithelial cell line HT-29. These observations may be relevant in the context of bacterial infection, as we demonstrated that anti-FliC response are protective against systemic infection by Salmonella typhimurium. Taken together, our study delineates a distinct co-operative and reciprocal interaction between the innate and adaptive arms of immunity in modulating their responses to FliC. more...

Iron is required to facilitate the redox activity of heme proteins expressed in both immune and non-immune cells. In this study, we investigated whether the chelation of iron by enterobactin (Ent; a prototypical bacterial siderophore) can affect the immune and nitrosative responses of RAW 264.7 and bone marrow-derived macrophages. We observed that Ent dose-dependently inhibited the LPS-induced secretion of cytokines (i.e., serum amyloid A, IL-6, lipocalin 2) and nitrite in the macrophages. The LPS-induced expression of inducible nitric oxide synthase (iNOS; a heme protein) was also inhibited by Ent at both the mRNA and protein levels. To demonstrate the physiological relevance of these findings, we next investigated whether Ent could protect the intracellular pathogen Salmonella enterica serovars typhimurium in the gentamycin protection assay. Ent reduced the nitrite levels in Salmonella-infected macrophages, but upregulated the expression of iNOS (mRNA, protein) and Arginase-1 (mRNA). More importantly, Ent-treated macrophages were impaired in mediating the killing of Salmonella. Ent-sufficient Salmonella also survived better compared with their isogenic Ent-deficient strains, whereas the addition of exogenous Ent substantially improved the survival of both strains. The inhibitory effects of Ent, however, were abrogated when saturated with iron (1:1 ratio), thus indicating that Ent is required to be in its iron-free form to mediate efficient inhibition on the macrophages. Taken together, our findings advance the concept that Ent not only facilitate bacterial iron uptake, but also serve to safeguard bacteria against macrophage immune responses. more...

Short-chain fatty acids (SCFAs), major microbial fermentation products of dietary fiber in the gut, are shown to improve gut health. We hypothesized that soluble fiber (inulin and pectin) which are more accessible for microbiota fermentation may offer more pronounced mucoprotection than insoluble fiber (cellulose) in a murine model of chronic colitis. WT (BL6) mice receiving dietary cellulose exhibited robust colitis upon IL-10R neutralization as examined by serological, biochemical, histological and immunological parameters. Similar results were observed in colitis-prone toll-like receptor 5 knockout (Tlr5KO) mice. Surprisingly, prebiotic fiber inulin failed to protect against colitis in both WT and Tlr5KO mice. Remarkably, pectin ameliorates colitis development. GC-MS analysis of cecal content revealed that cecal butyrate, an inflammasome (NLRP3) response modulator in the gut, was augmented in inulin-fed colitic mice. Serum cytokine analysis showed that dietary inulin elevated IL-18, but diminished the IL-1 receptor antagonist (sIL-1Ra). On the flip side, dietary pectin reduced IL-18 and augmented sIL-1Ra. More importantly, suppressing butyrate production by metronidazole, which specifically depletes the butyrate producers in the gut, protected the mice from chronic colitis, suggesting that SCFAs-inflammasome axis shapes the beneficial effects of dietary fiber on the gut health. Collectively, mitigation of chronic colitis by dietary pectin can be partly explained by reduced cecal butyrate and elevated IL-1β inhibitor, sIL-1Ra. A detailed mechanistic study on how pectin-derived metabolites modulate inflammasome signaling may yield development of novel therapeutics to treat intestinal inflammation. more...

Green tea (Camellia sinensis, Theaceae) contains a plethora of antioxidant polyphenols that may promote health. Amongst these polyphenols, the (−)-epigallocatechin-3-gallate (EGCG) is extensively studied for its potential therapeutic properties in models of inflammatory bowel disease (IBD), although its molecular mechanism is not completely understood. Here, we demonstrate that EGCG can inhibit the activity of myeloperoxidase (MPO; a pro-oxidant neutrophil enzyme associated with IBD flares) in a dose-dependent manner in vitro. By using spectral analysis, we show that EGCG prevents the MPO-catalyzed reaction by reverting the reactive peroxidase heme back to its native inactive ferric state. Oral administration of EGCG to dextran sodium sulfate (DSS)-induced colitic mice significantly reduced the colonic MPO activity and alleviated pro-inflammatory mediators associated with gut inflammation, confirming that our findings extend to in vivo conditions as well. In light of previous reports that EGCG can complex with innate immune protein lipocalin-2 and iron, we next tested whether these factors can affect the bioactivity of EGCG. Intriguingly, the presence of lipocalin-2 or iron significantly mitigate the ability of EGCG to inhibit MPO in vitro. Further, the efficacy of EGCG against DSS-induced gut inflammation is diminished when orally co-administered with iron. These findings indicate that EGCG-mediated inhibition of MPO could be one of the mechanisms by which EGCG exerts its mucoprotective effects. However, counter-regulatory factors such as host lipocalin-2 and dietary iron could explain why EGCG does not ameliorate gut inflammation in some IBD patients. more...

Natural dietary fiber serves as a substrate for gut microbiotal fermentation generating short chain fatty acids, which are thought to offer protection against inflammatory bowel disease (IBD). However, certain fibers are known to cause moderate to severe intestinal side effects in IBD patients. We hypothesize that soluble fibers (inulin & pectin) which are more accessible for microbiotal fermentation may offer pronounced gut protective effects than insoluble fiber (cellulose). To demonstrate this, chronic colitis was induced in dietary cellulose, inulin or pectin fed C57BL/6 mice by administering three weekly injections of α-IL-10R neutralizing antibody. Mice were euthanized two weeks after the last injection and colitis development was examined by serological, biochemical, histological and immunological parameters. WT mice receiving dietary cellulose along with α-IL-10R mAb exhibited robust colitis as characterized by splenomegaly, colomegaly, elevated colonic MPO activity, pro-inflammatory cytokines (Kc, Il-1β & iNos), systemic and fecal Lcn2 levels (an inflammatory marker) and colon histology score when compared to control mice. Similar results were observed in toll-like receptor 5 knockout (Tlr5KO) mice which are prone to develop microbiota-dependent spontaneous colitis. Surprisingly, prebiotic fiber inulin failed to protect against α-IL-10R neutralization-induced chronic colitis in both WT and Tlr5KO mice. Interestingly, pectin ameliorates colitis development, supporting the notion that not all dietary fibers are created equal in modulating host gut inflammation. Further mechanistic study on how pectin dampens gut inflammation may yield development of novel dietary therapeutics to treat human IBD. more...

The gut microbiota plays a key role in host metabolism. Toll-Like Receptor 5 (TLR5), a flagellin receptor, is required for gut microbiota homeostasis. Accordingly, TLR5 deficient (T5KO) mice are prone to develop microbiota-dependent spontaneous colitis and metabolic syndrome. Herein, we investigated whether metabolic syndrome in T5KO mice correlates with hepatic dyslipidemia and inflammation. T5KO mice displayed elevated neutral lipids with substantial enrichment of C18:1 (n9) relative to wild-type littermates. Oleate enrichment of hepatic lipids was microbiota-dependent. Analyzing cecal contents via 1H NMR-based metabolomics revealed that T5KO mice exhibited elevated short chain fatty acids (SCFA) with a concomitant increase in colonic SCFA receptors and hepatic pro-inflammatory genes and lipogenic enzymes including stearoyl-CoA desaturase1 (SCD1). SCFA treatment further aggravated metabolic syndrome in T5KO mice. Interestingly, deletion of hepatic SCD1 not only prevented hepatic neutral lipid oleate enrichment but also ameliorated hepatic inflammation and metabolic syndrome in T5KO mice. Collectively, these results underscore the key role of the gut microbiota-liver axis in the pathogenesis of metabolic diseases. more...

Fatty acids in lipid bilayer substantially influence membrane fluidity and thus dictate cellular functions. Stearoyl CoA Desaturase (SCD-1) is a lipogenic enzyme that assists in the de novo biosynthesis of oleate (C18:1, n9), a major fatty acid in the phospholipids of lipid bilayer. Accordingly SCD-1KO mice display substantially reduced oleate in cell membranes. We investigated the colitogenic capacity of effector T-cells from SCD-1KO mice and their WT littermates in adoptive T-cell transfer colitis model. Splenic effector T-cells (CD4+CD25-) from age and gender matched WT and SCD-1KO mice were isolated by cell sorting using magnetic beads and flow cytometry. RAG-1KO (n=5) were administered 5.0x105 CD4+CD25- cells/mouse intraperitoneally and monitored for body weights and colitis development. RAG-1KO mice which received CD4+CD25- cells from SCD-1KO mice displayed colomegaly, splenomegaly, elevated colonic, systemic lipocalin-2, a general inflammatory marker and hallmarks of colitis at histologic level. Interestingly, colonic TNFα and IL-10 gene transcripts were significantly reduced in SCD-1KO CD4+CD25- recipients whereas the opposite is true for iNOS and lipocalin-2. Further, systemic sIL-1Ra was significantly reduced in SCD-1KO CD4+CD25- recipients when compared to WT T cell recipients. Our results demonstrate that effector T-cells from oleate poor environment attain colitogenic properties and may be associated with increased immunoreactivity to gut microbiotal antigens. more...

During an inflammatory response in the gut, several commensal bacteria such as E.coli not only survive but also thrive and consequently contribute to the disease pathogenesis. The mechanisms by which such opportunistic pathogens bloom despite the hostile pro-inflammatory milieu of the inflamed gut remain largely unknown. We demonstrated that enterobactin (Ent), a siderophore released by E. coli, is a potent inhibitor of a key bactericidal enzyme of the host innate immune system, myeloperoxidase (MPO). The inhibitory activity is specific as glycosylated Ent (salmochelin) and non-catecholate siderophores such as yersiniabactin and ferrichrome failed to inhibit MPO activity. An E. coli ferrienterobactin permease mutant (ΔfepA) overexpressing Ent, but not 3-dehydroquinate synthase (ΔaroB)/ΔfepA double mutant inhibited MPO activity and exhibited enhanced survival in inflamed guts. This survival advantage was counter-regulated by the host siderophore binding protein, lipocalin 2, which rescued MPO from Ent-mediated inhibition. Spectral analysis revealed that Ent interferes with compound I [oxoiron, Fe(IV)=O] and reverts the enzyme back to its native ferric [Fe(III)] state. These findings define a fundamental mechanism by which E. coli surpasses the host innate immune responses during inflammatory gut diseases and gains a distinct survival advantage. more...

Apolipoprotein E (ApoE), a secreted protein, plays major role in cholesterol metabolism. ApoE is also known to exhibit anti-inflammatory properties. However, its role in the intestine, specifically during inflammation is not very clear. First, we verified upregulation of colonic ApoE transcripts in various models of murine acute and chronic colitis models. Age and gender matched ApoEKO mice and their WT littermates (n=5) were given either one or 4 weekly injections of IL-10 receptor (IL-10R) neutralizing mAb intraperitoneally and colitis development was monitored. ApoEKO mice given single injection of IL-10R mAb displayed acute colitis. Four weekly injections IL-10R mAb resulted in the development of chronic colitis as measured by colomegaly, splenomegaly and elevated colonic pro-inflammatory gene expression (TNFα, IFNγ) and histological score when compared to WT littermates. IL-10R neutralization also induced substantial levels of lipocalin 2, a general inflammatory marker in the feces, colon, and circulation in ApoEKO mice. Ablation of gut microbiota by broad-spectrum antibiotics substantially protected ApoEKO mice against IL-10R neutralization induced chronic colitis. In addition, cohousing ApoEKO mice with IL-10KO mice exaggerated spontaneous colitis (rectal prolapse) in IL-10KO mice when compared to WT suggesting ApoEKO harbor colitogenic microbiota. Collectively, our results demonstrate that ApoE plays a critical role in protecting the gut against inflammation. more...

Sepsis is characterized by hypoferremia, a primitive protective mechanism to limit iron-induced oxidative stress. Lipocalin-2 (Lcn2) which is highly upregulated during inflammation and has been shown to transport iron under physiologic conditions. Accordingly, we hypothesized that such upregulation of Lcn2 serves to protect against sepsis associated oxidative stress. Lcn2 deficient mice (Lcn2KO) and their WT littermates were given E. coli LPS (20mg/kg BW) i.p. and monitored for mortality. Serum TNFα, IL-18, LDH, aminotransferases, iron, glutathione and cysteine were analyzed. Splenocyte apoptosis was assayed by TUNEL, caspase 3 cleavage and flow cytometry. In WT mice, LPS-induced systemic Lcn2 peaked at 24h and returned to basal levels by 48h. All Lcn2KO died by day 8 while only 20% mortality was observed in WT mice. In addition, Lcn2KO exhibited substantial elevation of every parameter tested including both pro- and anti-inflammatory cytokines. Further,Lcn2KO splenocytes exhibited extensive apoptosis as measured by caspase 3 cleavage, TUNEL staining and flow cytometry. More importantly, when compared to WT mice, Lcn2KO mice failed to exhibit LPS-induced hypoferremia at early time points and exhibited severe hepatic oxidative stress. Our data demonstrate that Lcn2 facilitates LPS-induced hypoferremia and serves as a host protective factor by reducing oxidative stress. Given Lcn2 multifunctionality, it may be developed as a potential therapeutic agent to treat sepsis. more...

Lipocalin-2 (Lcn-2) is an innate immune protein whose expression is upregulated by several orders of magnitude in response to inflammation/injury/infection by variety of cells including neutrophils. However, the role of Lcn-2 induction in IC-induced inflammation is largely unknown. Here in, we have investigated the role of Lcn-2 during IC-mediated inflammation using Lcn-2 knock out (Lcn-2KO) and wild type (WT) mice. IC-mediated inflammation upregulated systemic Lcn-2 in WT mice when compared to naïve mice. The neutrophil chemoattractant KC was significantly elevated in WT, but not in Lcn-2KO mice, in response to IC-induced inflammation. Consistently Lcn-2KO mice showed about 50% reduced IC-mediated inflammation when compared to WT mice. Histopathological analysis of skin biopsy showed reduced inflammatory cell infiltration at inflamed site in Lcn-2KO mice relative to WT mice. Administration of recLcn-2 into Lcn-2KO mice resulted in inflammation similar to that of WT mice in response to IC. Lastly, we observed that neutralization of Lcn-2 via neutralizing mAb significantly reduced inflammation in WT mice. These results suggest that engagement of FcγRs expressed on local inflammatory cells with ICs leads to the induction of Lcn-2. Such IC-induced Lcn-2 expression may be essential for infiltration inflammatory cells at the site of inflammation. Our studies show that therapeutic targeting of Lcn-2 may be useful approach for treating IC-mediated inflammatory/autoimmune diseases more...