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Necrotizing enterocolitis (NEC) is the most devastating gastrointestinal disease of the premature infant. We have recently shown that NEC development occurs after an increase in proinflammatory CD4+Th17 (Th17) cells and reduced anti-inflammatory Foxp3+ regulatory T cells (Tregs) to the premature small intestine of mice and humans, which can be experimentally reversed in mice by administration of all-trans retinoic acid (ATRA). We have also shown that NEC is characterized by apoptosis of Lgr5-positive intestinal stem cells (ISCs–Lgr5+cells) within the crypts of Lieberkühn, which are subsequently essential for intestinal homeostasis. We now hypothesize that the normal lymphocyte balance within the lamina propria of the intestine can be achieved via administration of ATRA which restores mucosal integrity by preventing the loss of intestinal stem cells. Utilizing both in vivo and in vitro strategies we now demonstrate that Th17 recruitment and Treg depletion lead to increased apoptosis within ISC niches, significantly impairing proliferative capacity and mucosal healing. ATRA exerted its protective effects by preventing T cell imbalance, ultimately leading to the protection of the ISC pool preventing the development of NEC in mice. These findings raise the exciting possibility that dietary manipulations could prevent and treat NEC by modulating lymphocyte balance and the ISC pool within the newborn small intestine.

Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC1α) is the primary regulator of mitochondrial biogenesis and was recently found to be highly expressed within the intestinal epithelium. PGC1α is decreased in the intestinal epithelium of patients with inflammatory bowel disease, but its role in pathogenesis is uncertain. We now hypothesize that PGC1α protects against the development of colitis and helps to maintain the integrity of the intestinal barrier. We selectively deleted PGC1α from the intestinal epithelium of mice by breeding a PGC1α(loxP/loxP) mouse with a villin-cre mouse. Their progeny (PGC1α(ΔIEC) mice) were subjected to 2% dextran sodium sulfate (DSS) colitis for 7 days. The SIRT1 agonist SRT1720 was used to enhance PGC1α activation in wild-type mice during DSS exposure. Mice lacking PGC1α within the intestinal epithelium were more susceptible to DSS colitis than their wild-type littermates. Pharmacologic activation of PGC1α successfully ameliorated disease and restored mitochondrial integrity. These findings suggest that a depletion of PGC1α in the intestinal epithelium contributes to inflammatory changes through a failure of mitochondrial structure and function as well as a breakdown of the intestinal barrier, which leads to increased bacterial translocation. PGC1α induction helps to maintain mitochondrial integrity, enhance intestinal barrier function, and decrease inflammation.

Breast milk is the most effective strategy to protect infants against necrotizing enterocolitis (NEC), a devastating disease that is characterized by severe intestinal necrosis. Previous studies have demonstrated that the lipopolysaccharide receptor Toll-like receptor 4 (TLR4) plays a critical role in NEC development via deleterious effects on mucosal injury and repair. We now hypothesize that breast milk protects against NEC by inhibiting TLR4 within the intestinal epithelium, and sought to determine the mechanisms involved. Breast milk protected against NEC and reduced TLR4 signaling in wild-type neonatal mice, but not in mice lacking the epidermal growth factor receptor (EGFR), whereas selective removal of EGF from breast milk reduced its protective properties, indicating that breast milk inhibits NEC and attenuates TLR4 signaling via EGF/EGFR activation. Overexpression of TLR4 in the intestinal epithelium reversed the protective effects of breast milk. The protective effects of breast milk occurred via inhibition of enterocyte apoptosis and restoration of enterocyte proliferation. Importantly, in IEC-6 enterocytes, breast milk inhibited TLR4 signaling via inhibition of glycogen synthase kinase-3β (GSK3β). Taken together, these findings offer mechanistic insights into the protective role for breast milk in NEC, and support a link between growth factor and innate immune receptors in NEC pathogenesis.

The mechanisms that lead to the development of remote lung injury after trauma remain unknown, although a central role for the gut in the induction of lung injury has been postulated. We hypothesized that the development of remote lung injury after trauma/hemorrhagic shock requires activation of TLR4 in the intestinal epithelium, and we sought to determine the mechanisms involved. We show that trauma/hemorrhagic shock caused lung injury in wild-type mice, but not in mice that lack TLR4 in the intestinal epithelium, confirming the importance of intestinal TLR4 activation in the process. Activation of intestinal TLR4 after trauma led to increased endoplasmic reticulum (ER) stress, enterocyte apoptosis, and the release of circulating HMGB1, whereas inhibition of ER stress attenuated apoptosis, reduced circulating HMGB1, and decreased lung injury severity. Neutralization of circulating HMGB1 led to reduced severity of lung injury after trauma, and mice that lack HMGB1 in the intestinal epithelium were protected from the development of lung injury, confirming the importance of the intestine as the source of HMGB1, whose release of HMGB1 induced a rapid protein kinase C ζ–mediated internalization of surface tight junctions in the pulmonary epithelium. Strikingly, the use of a novel small-molecule TLR4 inhibitor reduced intestinal ER stress, decreased circulating HMGB1, and preserved lung architecture after trauma. Thus, intestinal epithelial TLR4 activation leads to HMGB1 release from the gut and the development of lung injury, whereas strategies that block upstream TLR4 signaling may offer pulmonary protective strategies after trauma.

Toxoplasma gondii is an obligate intracellular protozoan pathogen that traffics to the central nervous system (CNS) following invasion of its host. In the CNS, T. gondii undergoes transformation from a rapidly dividing tachyzoite to a long-lived, slow-dividing bradyzoite contained within cysts. The role of extracellular adenosine in T. gondii pathogenesis has not been previously investigated. T. gondii uses host purines such as adenosine for its energy needs, as it is unable to make its own. Here, we show that CD73 −/− mice, which lack the ability to generate extracellular adenosine, are protected from T. gondii chronic infection, with significantly fewer cysts and reduced susceptibility to reactivation of infection in the CNS independent of host effector function. Parasite dissemination to the brain was unimpaired in CD73 −/− hosts, suggesting that the reduced cyst number is due to impaired parasite differentiation in the CNS. Confirming this, T. gondii tachyzoites formed fewer cysts following alkaline pH stress in astrocytes isolated from CD73 −/− mice compared with wild type, and in fibroblasts treated with a CD73 inhibitor. Cyst formation was rescued in CD73 −/− astrocytes supplemented with adenosine, but not with adenosine receptor agonist 5′- N -ethylcarboxamidoadenosine. Furthermore, mice lacking adenosine receptors had no defect in cyst formation. Based on these findings, we conclude that CD73 expression promotes Toxoplasma bradyzoite differentiation and cyst formation by a mechanism dependent on the generation of adenosine, but independent of adenosine receptor signaling. Overall, these findings suggest that modulators of extracellular adenosine may be used to develop therapies aimed at defending against human toxoplasmosis.

The fetal intestinal mucosa is characterized by elevated Toll-like receptor 4 (TLR4) expression, which can lead to the development of necrotizing enterocolitis (NEC)—a devastating inflammatory disease of the premature intestine—upon exposure to microbes. To define endogenous strategies that could reduce TLR4 signaling, we hypothesized that amniotic fluid can inhibit TLR4 signaling within the fetal intestine and attenuate experimental NEC, and we sought to determine the mechanisms involved. We show here that microinjection of amniotic fluid into the fetal (embryonic day 18.5) gastrointestinal tract reduced LPS-mediated signaling within the fetal intestinal mucosa. Amniotic fluid is abundant in EGF, which we show is required for its inhibitory effects on TLR4 signaling via peroxisome proliferator-activated receptor, because inhibition of EGF receptor (EGFR) with cetuximab or EGF-depleted amniotic fluid blocked the inhibitory effects of amniotic fluid on TLR4, whereas amniotic fluid did not prevent TLR4 signaling in EGFR- or peroxisome proliferator-activated receptor γ–deficient enterocytes or in mice deficient in intestinal epithelial EGFR, and purified EGF attenuated the exaggerated intestinal mucosal TLR4 signaling in wild-type mice. Moreover, amniotic fluid-mediated TLR4 inhibition reduced the severity of NEC in mice through EGFR activation. Strikingly, NEC development in both mice and humans was associated with reduced EGFR expression that was restored upon the administration of amniotic fluid in mice or recovery from NEC in humans, suggesting that a lack of amniotic fluid-mediated EGFR signaling could predispose to NEC. These findings may explain the unique susceptibility of premature infants to the development of NEC and offer therapeutic approaches to this devastating disease.

Oral infection of certain inbred mouse strains with the protozoan Toxoplasma gondii triggers inflammatory pathology resembling lesions seen during human inflammatory bowel disease, in particular Crohn's disease (CD). Damage triggered by the parasite is largely localized to the distal portion of the small intestine, and as such is one of only a few models for ileal inflammation. This is important because ileal involvement is a characteristic of CD in over two-thirds of patients. The disease induced by Toxoplasma is mediated by Th1 cells and the cytokines tumor necrosis factor-α and interferon-γ. Inflammation is dependent upon IL-23, also identified by genome-wide association studies as a risk factor in CD. Development of lesions is concomitant with emergence of E. coli that display enhanced adhesion to the intestinal epithelium and subepithelial translocation. Furthermore, depletion of gut flora renders mice resistant to Toxoplasma-triggered ileitis. Recent findings suggest complex CCR2-dependent interactions between lamina propria T cells and intraepithelial lymphocytes in fueling proinflammatory pathology in the intestine. The advantage of the Toxoplasma model is that disease develops rapidly (within 7–10 days of infection) and can be induced in immunodeficient mice by adoptive transfer of mucosal T cells from infected donors. We propose that Toxoplasma acts as a trigger setting into motion a series of events culminating in loss of tolerance in the intestine and emergence of pathogenic T cell effectors. The Toxoplasma trigger model is providing new leaps in our understanding of immunity in the intestine.

Oral infection of C57BL/6 mice with Toxoplasma gondii triggers severe necrosis in the ileum within 7-10 days of infection. Lesion development is mediated by Th-1 cytokines, CD4+ T cells, and subepithelial bacterial translocation. As such, these features share similarity to Crohn's disease. Recently, we uncovered a role for intraepithelial lymphocytes (IELs) in mediating pathology after Toxoplasma infection. We show here that αβ and not γδ T-cell IELs mediate intestinal damage. By adoptive transfer of mucosal T cells into naive Rag1⁻/⁻ mice, we demonstrate that IELs do not function alone to cause inflammatory lesions, but act with CD4+ T lymphocytes from the lamina propria (LP). Furthermore, recipient mice pretreated with broad-spectrum antibiotics to eliminate intestinal flora resisted intestinal disease after transfer of IELs and LP lymphocytes. Our data provide valuable new insights into the mechanisms of intestinal inflammation, findings that have important implications for understanding human inflammatory bowel disease.

Neutrophils play a major role in the innate immune system and are normally considered to be short-lived effector cells that exert anti-microbial activity and sometimes immunopathology. Here, we show that these cells possess an additional function as professional antigen-presenting cells capable of priming a T(h)1- and T(h)17-acquired immune response. Using flow cytometry, fluorescence microscopy and western blotting, we show that mouse neutrophils express MHC class II and co-stimulatory molecules CD80 and CD86 after T-cell co-incubation. Neutrophils pulsed with ovalbumin (OVA) process and present peptide antigen to OVA-specific T cells in an MHC class II-dependent manner. Importantly, we demonstrate that neutrophils can prime antigen-specific T(h)1 and T(h)17 immune responses even without the addition of exogenous cytokines to cell cultures.

Mice of the C57BL/6 strain develop acute ileal inflammation after infection with the protozoan parasite Toxoplasma gondii. This pathology resembles many key features of human Crohn's disease, including a Th1 cytokine profile with high levels of interferon gamma (IFN-gamma), interleukin 12 (IL)-12, and tumor necrosis factor alpha (TNF)-alpha, presence of pathogenic CD4(+) T cells, and infiltration of gut flora into inflammed tissue. Using CCR2(-/-) mice, we identify a role for this chemokine receptor in the pathogenesis of inflammatory pathology during T. gondii infection. Lack of chemokine (C-C motif) receptor 2 (CCR2) was associated with low levels of CD103(+) T lymphocytes in the intraepithelial compartment, Peyer's patch, and lamina propria relative to wild-type animals. Adoptive transfer of wild-type, but not IFN-gamma(-/-), intraepithelial T lymphocytes converted CCR2 knockout mice from a resistant to susceptible phenotype with respect to parasite-triggered inflammatory gut pathology. These results for the first time show a role for intraepithelial T lymphocytes in pathogenesis of ileitis triggered by a microbial pathogen.

TLR adaptor MyD88 activation is important in host resistance to Toxoplasma gondii during i.p. infection, but the function of this signaling pathway during oral infection, in which mucosal immunity assumes a predominant role, has not been examined. In this study, we show that MyD88−/− mice fail to control the parasite and succumb within 2 wk of oral infection. Early during infection, T cell IFN-γ production, recruitment of neutrophils and induction of p47 GTPase IGTP (Irgm3) in the intestinal mucosa were dependent upon functional MyD88. Unexpectedly, these responses were MyD88-independent later during acute infection. In particular, CD4+ T cell IFN-γ reached normal levels independently of MyD88, despite continued absence of IL-12 in these animals. The i.p. vaccination of MyD88−/− mice with an avirulent T. gondii uracil auxotroph elicited robust IFN-γ responses and protective immunity to challenge with a high virulence T. gondii strain. Our results demonstrate that MyD88 is required to control Toxoplasma infection, but that the parasite can trigger adaptive immunity without the need for this TLR adaptor molecule.

The murine cell surface determinant Gr-1 is expressed at high level on neutrophils. Depletion of polymorphonuclear leukocytes with anti-Gr-1(+) monoclonal antibody results in increased susceptibility and dysregulated immunity to many microbial pathogens, a finding widely interpreted to indicate the importance of neutrophils during infection. Yet, in recent years it has become clear that additional cell types express the Gr-1 determinant, including dendritic cell and monocyte subpopulations. In this review, we evaluate current knowledge on the functional aspects of Gr-1(+) cell populations. We focus on infection with the opportunistic protozoan Toxoplasma gondii, a case where host survival depends on an intact Gr-1(+) cell population.

The nature and role of the intestinal leukocytes in necrotizing enterocolitis (NEC), a severe disease affecting premature infants, remain unknown. We now show that the intestine in mouse and human NEC is rich in lymphocytes that are required for NEC development, as recombination activating gene 1–deficient (Rag1–/–) mice were protected from NEC and transfer of intestinal lymphocytes from NEC mice into naive mice induced intestinal inflammation. The intestinal expression of the lipopolysaccharide receptor TLR4, which is higher in the premature compared with full-term human and mouse intestine, is required for lymphocyte influx through TLR4-mediated upregulation of CCR9/CCL25 signaling. TLR4 also mediates a STAT3-dependent polarization toward increased proinflammatory CD3+CD4+IL-17+ and reduced tolerogenic Foxp3+ Treg lymphocytes (Tregs). Th17 lymphocytes were required for NEC development, as inhibition of STAT3 or IL-17 receptor signaling attenuated NEC in mice, while IL-17 release impaired enterocyte tight junctions, increased enterocyte apoptosis, and reduced enterocyte proliferation, leading to NEC. Importantly, TLR4-dependent Th17 polarization could be reversed by the enteral administration of retinoic acid, which induced Tregs and decreased NEC severity. These findings identify an important role for proinflammatory lymphocytes in NEC development via intestinal epithelial TLR4 that could be reversed through dietary modification.

Gammadelta T cells play important but poorly defined roles in pathogen-induced immune responses and in preventing chronic inflammation and pathology. A major obstacle to defining their function is establishing the degree of functional redundancy and heterogeneity among gammadelta T cells. Using mice deficient in Vgamma1+ T cells which are a major component of the gammadelta T cell response to microbial infection, a specific immunoregulatory role for Vgamma1+ T cells in macrophage and gammadelta T cell homeostasis during infection has been established. By contrast, Vgamma1+ T cells play no significant role in pathogen containment or eradication and cannot protect mice from immune-mediated pathology. Pathogen-elicited Vgamma1+ T cells also display different functional characteristics at different stages of the host response to infection that involves unique and different populations of Vgamma1+ T cells. These findings, therefore, identify distinct and nonoverlapping roles for gammadelta T cell subsets in infection and establish the complexity and adaptability of a single population of gammadelta T cells in the host response to infection that is not predetermined, but is, instead, shaped by environmental factors.

The cellular cues that regulate the apoptosis of intestinal stem cells (ISCs) remain incompletely understood, yet may play a role in diseases characterized by ISC loss including necrotizing enterocolitis (NEC). Toll-like receptor-4 (TLR4) was recently found to be expressed on ISCs, where its activation leads to ISC apoptosis through mechanisms that remain incompletely explained. We now hypothesize that TLR4 induces endoplasmic reticulum (ER) stress within ISCs, leading to their apoptosis in NEC pathogenesis, and that high ER stress within the premature intestine predisposes to NEC development. Using transgenic mice and cultured enteroids, we now demonstrate that TLR4 induces ER stress within Lgr5 (leucine-rich repeat-containing G-protein-coupled receptor 5)-positive ISCs, resulting in crypt apoptosis. TLR4 signaling within crypts was required, because crypt ER stress and apoptosis occurred in TLR4(ΔIEC-OVER) mice expressing TLR4 only within intestinal crypts and epithelium, but not TLR4(ΔIEC) mice lacking intestinal TLR4. TLR4-mediated ER stress and apoptosis of ISCs required PERK (protein kinase-related PKR-like ER kinase), CHOP (C/EBP homologous protein), and MyD88 (myeloid differentiation primary response gene 88), but not ATF6 (activating transcription factor 6) or XBP1 (X-box-binding protein 1). Human and mouse NEC showed high crypt ER stress and apoptosis, whereas genetic inhibition of PERK or CHOP attenuated ER stress, crypt apoptosis, and NEC severity. Strikingly, using intragastric delivery into fetal mouse intestine, prevention of ER stress reduced TLR4-mediated ISC apoptosis and mucosal disruption. These findings identify a novel link between TLR4-induced ER stress and ISC apoptosis in NEC pathogenesis and suggest that increased ER stress within the premature bowel predisposes to NEC development.

Chemokines and their receptors play a critical role in orchestrating immunity to microbial pathogens, including the orally acquired Th1-inducing protozoan parasite Toxoplasma gondii. Chemokine receptor CXCR3 is associated with Th1 responses, and here we use bicistronic CXCR3-eGFP knock-in reporter mice to demonstrate upregulation of this chemokine receptor on CD4+ and CD8+ T lymphocytes during Toxoplasma infection. We show a critical role for CXCR3 in resistance to the parasite in the intestinal mucosa. Absence of the receptor in Cxcr3−/− mice resulted in selective loss of ability to control T. gondii specifically in the lamina propria compartment. CD4+ T cells were impaired both in their recruitment to the intestinal lamina propria and in their ability to secrete IFN-γ upon stimulation. Local recruitment of CD11b+Ly6C/G+ inflammatory monocytes, recently reported to be major anti-Toxoplasma effectors in the intestine, was not impacted by loss of CXCR3. However, inflammatory monocyte activation status, as measured by dual production of TNF-α and IL-12, was severely impaired in Cxcr3−/− mice. Strikingly, adoptive transfer of wild-type but not Ifnγ−/− CD4+ T lymphocytes into Cxcr3−/− animals prior to infection corrected the defect in inflammatory macrophage activation, simultaneously reversing the susceptibility phenotype of the knockout animals. Our results establish a central role for CXCR3 in coordinating innate and adaptive immunity, ensuring generation of Th1 effectors and their trafficking to the frontline of infection to program microbial killing by inflammatory monocytes., Author Summary Inflammatory monocytes have recently emerged as important effectors in intestinal defense against enteric pathogens, but requirements for their activation are poorly defined. Here we use the protozoan Toxoplasma gondii, an orally acquired Th1-inducing pathogen, to study the requirements for inflammatory macrophage activation in the intestinal mucosa. We find that CD4+ T lymphocytes, recruited in dependence upon their expression of the chemokine receptor CXCR3, mediate activation of intestinal mucosa inflammatory monocytes via secretion of IFN-γ, in turn resulting in control of infection. Thus, CXCR3 functions as a critical lynchpin coordinating anti-microbial communication between T lymphocytes and inflammatory monocyte effectors.

Necrotizing enterocolitis (NEC) is a devastating disease of premature infants characterized by severe intestinal necrosis and for which breast milk represents the most effective protective strategy. Previous studies have revealed a critical role for the lipopolysaccharide receptor toll-like receptor 4 (TLR4) in NEC development through its induction of mucosal injury, yet the reasons for which intestinal ischemia in NEC occurs in the first place remain unknown. We hypothesize that TLR4 signaling within the endothelium plays an essential role in NEC development by regulating perfusion to the small intestine via the vasodilatory molecule endothelial nitric oxide synthase (eNOS). Using a unique mouse system in which we selectively deleted TLR4 from the endothelium, we now show that endothelial TLR4 activation is required for NEC development and that endothelial TLR4 activation impairs intestinal perfusion without effects on other organs and reduces eNOS expression via activation of myeloid differentiation primary response gene 88. NEC severity was significantly increased in eNOS −/− mice and decreased upon administration of the phosphodiesterase inhibitor sildenafil, which augments eNOS function. Strikingly, compared with formula, human and mouse breast milk were enriched in sodium nitrate—a precursor for enteral generation of nitrite and nitric oxide—and repletion of formula with sodium nitrate/nitrite restored intestinal perfusion, reversed the deleterious effects of endothelial TLR4 signaling, and reduced NEC severity. These data identify that endothelial TLR4 critically regulates intestinal perfusion leading to NEC and reveal that the protective properties of breast milk involve enhanced intestinal microcirculatory integrity via augmentation of nitrate–nitrite–NO signaling.

Antimicrobial proteins influence intestinal microbial ecology and limit proliferation of pathogens, yet the regulation of their expression has only been partially elucidated. Here, we have identified a putative pathway involving epithelial cells and intestinal intraepithelial lymphocytes (iIELs) that leads to antimicrobial protein (AMP) production by Paneth cells. Mice lacking γδ iIELs (TCRδ-/-) express significantly reduced levels of the AMP angiogenin 4 (Ang4). These mice were also unable to up-regulate Ang4 production following oral challenge by Salmonella, leading to higher levels of mucosal invasion compared to their wild type counterparts during the first 2 hours post-challenge. The transfer of γδ iIELs from wild type (WT) mice to TCRδ-/- mice restored Ang4 production and Salmonella invasion levels were reduced to those obtained in WT mice. The ability to restore Ang4 production in TCRδ-/- mice was shown to be restricted to γδ iIELs expressing Vγ7-encoded TCRs. Using a novel intestinal crypt co-culture system we identified a putative pathway of Ang4 production initiated by exposure to Salmonella, intestinal commensals or microbial antigens that induced intestinal epithelial cells to produce cytokines including IL-23 in a TLR-mediated manner. Exposure of TCR-Vγ7+ γδ iIELs to IL-23 promoted IL-22 production, which triggered Paneth cells to secrete Ang4. These findings identify a novel role for γδ iIELs in mucosal defence through sensing immediate epithelial cell cytokine responses and influencing AMP production. This in turn can contribute to the maintenance of intestinal microbial homeostasis and epithelial barrier function, and limit pathogen invasion. © 2013 Walker et al.

Non-alcoholic fatty liver disease (NAFLD) is a common disease with a spectrum of presentations. The current study utilized a lithogenic diet model of NAFLD. The diet was fed to mice that are either resistant (AKR) or susceptible (BALB/c and C57BL/6) to hepatitis followed by molecular and flow cytometric analysis. Following this, a similar approach was taken in congenic mice with specific mutations in immunological genes. The initial study identified a significant and profound increase in multiple ligands for the chemokine receptor CCR2 and an increase in CD44 expression in susceptible C57BL/6 (B6) but not resistant AKR mice. Ccr2(-/-) mice were completely protected from hepatitis and Cd44(-/-) mice were partially protected. Despite protection from inflammation, both strains displayed similar histological steatosis scores and significant increases in serum liver enzymes. CD45(+)CD44(+) cells bound to hyaluronic acid (HA) in diet fed B6 mice but not Cd44(-/-) or Ccr2(-/-) mice. Ccr2(-/-) mice displayed a diminished HA binding phenotype most notably in monocytes, and CD8(+) T-cells. In conclusion, this study demonstrates that absence of CCR2 completely and CD44 partially reduces hepatic leukocyte recruitment. These data also provide evidence that there are multiple redundant CCR2 ligands produced during hepatic lipid accumulation and describes the induction of a strong HA binding phenotype in response to LD feeding in some subsets of leukocytes from susceptible strains.

Factors regulating the proliferation and apoptosis of intestinal stem cells (ISCs) remain incompletely understood. Because ISCs exist among microbial ligands, immune receptors such as toll-like receptor 4 (TLR4) could play a role. We now hypothesize that ISCs express TLR4 and that the activation of TLR4 directly on the intestinal stem cells regulates their ability to proliferate or to undergo apoptosis. Using flow cytometry and fluorescent in situ hybridization for the intestinal stem cell marker Lgr5, we demonstrate that TLR4 is expressed on the Lgr5-positive intestinal stem cells. TLR4 activation reduced proliferation and increased apoptosis in ISCs both in vivo and in ISC organoids, a finding not observed in mice lacking TLR4 in the Lgr5-positive ISCs, confirming the in vivo significance of this effect. To define molecular mechanisms involved, TLR4 inhibited ISC proliferation and increased apoptosis via the p53-up-regulated modulator of apoptosis (PUMA), as TLR4 did not affect crypt proliferation or apoptosis in organoids or mice lacking PUMA. In vivo effects of TLR4 on ISCs required TIR-domain-containing adapter-inducing interferon-β (TRIF) but were independent of myeloid-differentiation primary response-gene 88 (MYD88) and TNFα. Physiological relevance was suggested, as TLR4 activation in necrotizing enterocolitis led to reduced proliferation and increased apoptosis of the intestinal crypts in a manner that could be reversed by inhibition of PUMA, both globally or restricted to the intestinal epithelium. These findings illustrate that TLR4 is expressed on ISCs where it regulates their proliferation and apoptosis through activation of PUMA and that TLR4 regulation of ISCs contributes to the pathogenesis of necrotizing enterocolitis.

IL-12–mediated type 1 inflammation confers host protection against the parasitic protozoan Toxoplasma gondii. However, production of IFN-γ, another type 1 inflammatory cytokine, also drives lethality from excessive injury to the intestinal epithelium. As mechanisms that restore epithelial barrier function following infection remain poorly understood, this study investigated the role of trefoil factor 2 (TFF2), a well-established regulator of mucosal tissue repair. Paradoxically, TFF2 antagonized IL-12 release from dendritic cells (DCs) and macrophages, which protected TFF2-deficient (TFF2−/−) mice from T. gondii pathogenesis. Dysregulated intestinal homeostasis in naive TFF2−/− mice correlated with increased IL-12/23p40 levels and enhanced T cell recruitment at baseline. Infected TFF2−/− mice displayed low rates of parasite replication and reduced gut immunopathology, whereas wild-type (WT) mice experienced disseminated infection and lethal ileitis. p38 MAPK activation and IL-12p70 production was more robust from TFF2−/−CD8+ DC compared with WT CD8+ DC and treatment of WT DC with rTFF2 suppressed TLR-induced IL-12/23p40 production. Neutralization of IFN-γ and IL-12 in TFF2−/− animals abrogated resistance shown by enhanced parasite replication and infection-induced morbidity. Hence, TFF2 regulated intestinal barrier function and type 1 cytokine release from myeloid phagocytes, which dictated the outcome of oral T. gondii infection in mice.

Summary Toll-like receptor (TLR)/MyD88 signalling has emerged as a major pathway of pathogen recognition in the innate immune system. Here, we review recent data that begin to show how this pathway controls the immune response to protozoan infection, with particular emphasis on the opportunistic pathogen Toxoplasma gondii. The various ways that the parasite activates and suppresses TLR/MyD88 signalling defines several key principals that illuminate the complexities of the host–pathogen interaction. We also speculate how TLR/MyD88 signalling might be exploited to provide protection against Toxoplasma, as well as other protozoa and infection in general.

Macrophages infected with the opportunistic protozoan Toxoplasma gondii are unable to up-regulate many proinflammatory cytokine genes, including TNF (TNF-α), upon stimulation with LPS and other TLR ligands. In this study, we examined the influence of T. gondii on transcription factors associated with TNF-α transcription, as well as phosphorylation and acetylation of histone H3 at distal and proximal regions of the TNF-α promoter. During LPS stimulation, we found that Toxoplasma blocks nuclear accumulation of transcription factor c-Jun, but not that of cAMP response element-binding protein or NF-κB. However, chromatin immunoprecipitation studies revealed that binding of all of these transcription factors to the TNF promoter was decreased by T. gondii infection. Furthermore, the parasite blocked LPS-induced Ser10 phosphorylation and Lys9/Lys14 acetylation of histone H3 molecules associated with distal and proximal regions of the TNF-α promoter. Our results show that Toxoplasma inhibits TNF-α transcription by interfering with chromatin remodeling events required for transcriptional activation at the TNF promoter, revealing a new mechanism by which a eukaryotic pathogen incapacitates proinflammatory cytokine production during infection.

The MAPK family member JNK/stress-activated MAPK (SAPK) is involved in extracellular stress and proinflammatory cytokine responses, including production of cytokines such as IL-12. The JNK1 and 2 isoforms are widely expressed, but JNK3 is largely restricted to tissues of the brain, testis, and heart. In this study, we focus on mouse neutrophils, a cell type in which JNK/SAPK expression and activity has been given little study. We used Western blot analysis to examine expression patterns of JNK/SAPK in wild-type and JNK2−/− polymorphonuclear leukocytes (PMN). Surprisingly, neutrophils displayed a major deficiency in JNK1 expression, in contrast to macrophages that expressed high levels of both JNK1 and JNK2 MAPK. JNK1 expression was steadily reduced during the neutrophil maturation in bone marrow. We used PMN infection with the protozoan parasite Toxoplasma gondii to determine whether neutrophil JNK2 was functional. The parasite induced rapid JNK2 phosphorylation and intracellular FACS staining demonstrated preferential activation in infected neutrophils. Use of JNK2−/− neutrophils revealed that this MAPK family member was required for PMN IL-12p40 and CCL2/MCP-1 production. The chemotactic response displayed a minor JNK2 dependence but phagocytosis and oxidative burst activity did not require this MAPK. These findings are important because they demonstrate 1) a previously unrecognized unusual JNK expression pattern in mouse neutrophils, 2) JNK2 in PMN is activated by Toxoplasma invasion, and 3) a requirement for JNK2 in PMN IL-12p40 and CCL2/MCP-1 production in response to a microbial pathogen.

Background & Aims: Intestinal epithelial integrity and permeability is dependent on intercellular tight junction (TJ) complexes. How TJ integrity is regulated remains unclear, although phosphorylation and dephosphorylation of the integral membrane protein occludin is an important determinant of TJ formation and epithelial permeability. We have investigated the role intestinal intraepithelial lymphocytes (iIELs) play in regulating epithelial permeability in response to infection. Methods: Recombinant strains of Toxoplasma gondii were used to assess intestinal epithelial barrier function and TJ integrity in mice with intact or depleted populations of iIELs. Alterations in epithelial permeability were correlated with TJ structure and the state of phosphorylation of occludin. iIEL in vivo reconstitution experiments were used to identify the iIELs required to maintain epithelial permeability and TJ integrity. Results: In the absence of γδ + iIELs, intestinal epithelial barrier function and the ability to restrict epithelial transmigration of Toxoplasma and the unrelated intracellular bacterial pathogen Salmonella typhimurium was severely compromised. Leaky epithelium in γδ + iIEL-deficient mice was associated with the absence of phosphorylation of serine residues of occludin and lack of claudin 3 and zona occludens-1 proteins in TJ complexes. These deficiencies were attributable to the absence of a single subset of γδ T-cell receptor (TCR-Vγ7 + ) iIELs that, after reconstituting γδ iIEL-deficient mice, restored epithelial barrier function and TJ complexes, resulting in increased resistance to infection. Conclusions: These findings identify a novel role for γδ + iIELs in maintaining TJ integrity and epithelial barrier function that have implications for understanding the pathogenesis of intestinal inflammatory diseases associated with disruption of TJ complexes.

Gammadelta T cells play important but poorly defined roles in pathogen-induced immune responses and in preventing chronic inflammation and pathology. A major obstacle to defining their function is establishing the degree of functional redundancy and heterogeneity among gammadelta T cells. Using mice deficient in Vgamma1+ T cells which are a major component of the gammadelta T cell response to microbial infection, a specific immunoregulatory role for Vgamma1+ T cells in macrophage and gammadelta T cell homeostasis during infection has been established. By contrast, Vgamma1+ T cells play no significant role in pathogen containment or eradication and cannot protect mice from immune-mediated pathology. Pathogen-elicited Vgamma1+ T cells also display different functional characteristics at different stages of the host response to infection that involves unique and different populations of Vgamma1+ T cells. These findings, therefore, identify distinct and nonoverlapping roles for gammadelta T cell subsets in infection and establish the complexity and adaptability of a single population of gammadelta T cells in the host response to infection that is not predetermined, but is, instead, shaped by environmental factors.

G A A b st ra ct s Hypothesis: We hypothesized that LPS increases the intestinal Treg population in mice. Methods: Total spleen cells or purified splenic CD3+ T cells from C57BL/6 mice were stimulated in vitro with anti-CD3/28 and TGFβ to promote Treg generation. Parallel cultures were performed with additional LPS. Four days later, cells were stained for CD3, FoxP3 and helios and analyzed by flow cytometry. To analyze the effect of intestinal LPS on intestinal Treg population in vivo, germ-free C57BL/6 mice were administered ultrapure LPS by daily gastric gavage for 3 or 4 weeks. Mesenteric lymph node cells from LPS treated or untreated germ-free mice were analyzed for CD3, FoxP3 and helios expression. Last, peripheral T cells from conventional flora C57BL/6 WT, C57BL/10 WT and C57BL/10ScN (TLR4 deficient) mice kept under pathogen-free conditions were analyzed for Treg subpopulations. Results: The addition of LPS to anti-CD3/28 and TGF β stimulated, purified CD3 T cell cultures increased the ratio of helios negative Tregs to total Tregs from 43±19% to 89±6 % (N=2; p=0.02). Addition of LPS to total spleen cells stimulated with anti-CD3/28 in the presence of TGFβ increased the percentage of helios negative Tregs among total Tregs from 20±5 % to 40±2 % (N=3; p,0.01). In vivo, in a germ-free environment devoid of gut microbiota, orally administered LPS increased the total number of Treg cells 3.4-fold (p ,0.01; at least 2 experiments for each group). The percentage of helios negative Tregs (iTregs) also increased from 15±2% to 32±3% after 3 or 4 week of LPS treatment (p,0.001). Conversely, in animals populated with intestinal microbiota and maintained under pathogen-free conditions, the percentage of helios negative iTregs was decreased from 27±4% in C57BL/6 WT and 24±3% in C57BL/10 WT to 17±3% in C57BL/10 ScN TLR4 deficient mice (at least two experiments for each animal group; p,0.05 between C57BL/6 WT and TLR4 deficient as well as between C57BL/10 WT and TLR4 deficient). Conclusions: These findings support an important role for LPS-triggered TLR4 signaling in intestinal mucosal and peripheral Treg development.

RESULTS: Interestingly, initial examination of specimens (N 22) failed to correlate lack of feeding and TER(p 0.09). However, in fed samples, TER increased with patient age (p 0.01). Therefore results were then stratified by patient’s age. Fully fed bowel had significantly higher TER versus unfed bowel in age 10y.o.(p 0.05). This trend was also observed in infants (p 0.053). These changes in TER correlated with permeation of FITC-Dextran (p 0.03). We next correlated Ussing findings to immunofluorescence staining. Dramatic declines in staining of ZO-1, occludin, E-cadherin and Claudin-1 were observed in unfed segments. E-cadherin (p 0.01) and Claudin-4 (p 0.02) mRNA increased significantly in those samples with higher TER. ZO-1, Claudin-5 and occludin mRNA trended in a similar fashion, but not significantly.