Mast Cells Play No Role in the Pathogenesis of Postoperative Ileus Induced by Intestinal Manipulation

Introduction

Intestinal manipulation (IM) during abdominal surgery results in intestinal inflammation leading to hypomotility or ileus. Mast cell activation is thought to play a crucial role in the pathophysiology of postoperative ileus (POI). However, this conclusion was mainly drawn using mast cell-deficient mouse models with abnormal Kit signaling. These mice also lack interstitial cells of Cajal (ICC) resulting in aberrant gastrointestinal motility even prior to surgery, compromising their use as model to study POI. To avoid these experimental weaknesses we took advantage of a newly developed knock-in mouse model, Cpa3^Cre/+, devoid of mast cells but with intact Kit signaling.

Design

The role of mast cells in the development of POI and intestinal inflammation was evaluated assessing gastrointestinal transit and muscularis externa inflammation after IM in two strains of mice lacking mast cells, i.e. Kit^W-sh/W-sh and Cpa3^Cre/+ mice, and by use of the mast cell stabilizer cromolyn.

Results

Kit^W-sh/W-sh mice lack ICC networks and already revealed significantly delayed gastrointestinal transit even before surgery. IM did not further delay intestinal transit, but induced infiltration of myeloperoxidase positive cells, expression of inflammatory cytokines and recruitment of monocytes and neutrophils into the muscularis externa. On the contrary, Cpa3^Cre/+ mice have a normal network of ICC and normal gastrointestinal. Surprisingly, IM in Cpa3^Cre/+ mice caused delay in gut motility and intestinal inflammation as in wild type littermates mice (Cpa3^+/+). Furthermore, treatment with the mast cell inhibitor cromolyn resulted in an inhibition of mast cells without preventing POI.

Conclusions

Here, we confirm that IM induced mast cell degranulation. However, our data demonstrate that mast cells are not required for the pathogenesis of POI in mice. Although there might be species differences between mouse and human, our results argue against mast cell inhibitors as a therapeutic approach to shorten POI.     

Altered transit and bacterial overgrowth in the cystic fibrosis mouse small intestine

Small intestinal bacterial overgrowth (SIBO) may play an important role in the gastrointestinal complications of cystic fibrosis (CF). This work explored two potential factors in development of SIBO in the CF (cftr(tm1UNC)) mouse: impaired Paneth cell innate defenses and altered gastrointestinal motility. Postnatal differentiation of Paneth cells was followed by Defcr, Lyzs, and Ang4 gene expression, and SIBO was measured by quantitative PCR of the bacterial 16S rRNA gene. Paneth cell gene expression was low in 4-day-old CF and wild-type (WT) mice and increased similarly in both groups of mice between 12 and 16 days. Peak Paneth cell gene expression was reached by 40 days of age and was less for Defcr and Lyzs in CF mice compared with WT, whereas Ang4 levels were greater in CF mice. SIBO occurred by postnatal day 8 in CF mice, which is before Paneth cell development. With the use of gavaged rhodamine-dextran to follow motility, gastric emptying in CF mice was slightly decreased compared with WT, and small intestinal transit was dramatically less. Since antibiotics improve weight gain in CF mice, their effects on gastric emptying and small intestinal transit were determined. Antibiotics did not affect gastric emptying or transit in CF mice but did significantly slow intestinal transit in WT mice, suggesting a potential role of normal microflora in regulating transit. In conclusion, small intestinal transit was significantly slower in CF mice, and this is likely a major factor in SIBO in CF.     

The Spleen Responds to Intestinal Manipulation but Does Not Participate in the Inflammatory Response in a Mouse Model of Postoperative Ileus

Background

Postoperative ileus is characterized by a transient impairment of the gastrointestinal motility after abdominal surgery. The intestinal inflammation, triggered by handling of the intestine, is the main factor responsible for the prolonged dysmotility of the gastrointestinal tract. Secondary lymphoid organs of the intestine were identified as essential components in the dissemination of inflammation to the entire gastrointestinal tract also called field effect. The involvement of the spleen, however, remains unclear.

Aim

In this study, we investigated whether the spleen responds to manipulation of the intestine and participates in the intestinal inflammation underlying postoperative ileus.

Methods

Mice underwent Laparotomy (L) or Laparotomy followed by Intestinal Manipulation (IM). Twenty-four hours later, intestinal and colonic inflammation was assessed by QPCR and measurement of the intestinal transit was performed. Analysis of homeostatic chemokines in the spleen was performed by QPCR and splenic cell populations analysed by Flow Cytometry. Blockade of the egress of cells from the spleen was performed by administration of the Sphingosine-1-phosphate receptor 1 (S1P₁) agonist CYM-5442 10 h after L/IM.

Results

A significant decrease in splenic weight and cellularity was observed in IM mice 24 h post-surgery, a phenomenon associated with a decreased splenic expression level of the homeostatic chemokine CCL19. Splenic denervation restored the expression of CCL19 and partially prevented the reduction of splenocytes in IM mice. Treatment with CYM-5442 prevented the egress of splenocytes but did not ameliorate the intestinal inflammation underlying postoperative ileus.

Conclusions

Intestinal manipulation results in two distinct phenomena: local intestinal inflammation and a decrease in splenic cellularity. The splenic response relies on an alteration of cell trafficking in the spleen and is partially regulated by the splenic nerve. The spleen however does not participate in the intestinal inflammation during POI.     

Acute intratracheal Pseudomonas aeruginosa infection in cystic fibrosis mice is age-independent

Background

Since the discovery of the human CFTR gene in 1989 various mouse models for cystic fibrosis (CF) have been generated and used as a very suitable and popular tool to approach research on this life-threatening disease. Age related changes regarding the course of disease and susceptibility towards pulmonary infections have been discussed in numerous studies.

Methods

Here, we investigated Cftr^{TgH(neoim)Hgu} and Cftr^tm1Unc-Tg(FABPCFTR)1Jaw/J CF mice and their non-CF littermates during an acute lung infection with Pseudomonas aeruginosa for age dependent effects of their lung function and immune response.Mice younger than three or older than six months were intratracheally infected with P. aeruginosa TBCF10839. The infection was monitored by lung function of the animals using non-invasive head-out spirometry and the time course of physiological parameters over 192 hours. Quantitative bacteriology and lung histopathology of a subgroup of animals were used as endpoint parameters.

Results

Age-dependent changes in lung function and characteristic features for CF like a shallower, faster breathing pattern were observed in both CF mouse models in uninfected state. In contrast infected CF mice did not significantly differ from their non-CF littermates in susceptibility and severity of lung infection in both mouse models and age groups. The transgenic Cftr^tm1Unc-Tg(FABPCFTR)1Jaw/J and their non-CF littermates showed a milder course of infection than the Cftr^{TgH(neoim)Hgu} CF and their congenic C57Bl/6J non-CF mice suggesting that the genetic background was more important for outcome than Cftr dysfunction.

Conclusions

Previous investigations of the same mouse lines have shown a higher airway susceptibility of older CF mice to intranasally applied P. aeruginosa. The different outcome of intranasal and intratracheal instillation of bacteria implies that infected CF epithelium is impaired during the initial colonization of upper airways, but not in the subsequent response of host defense.     

Roles of mast cells in the pathogenesis of inflammatory myopathy

Introduction

In addition to the pivotal roles of mast cells in allergic diseases, recent data suggest that mast cells play crucial roles in a variety of autoimmune responses. However, their roles in the pathogenesis of autoimmune skeletal muscle diseases have not been clarified despite their distribution in skeletal muscle. Therefore, the objective of this study is to determine the roles of mast cells in the development of autoimmune skeletal muscle diseases.

Methods

The number of mast cells in the affected muscle was examined in patients with dermatomyositis (DM) or polymyositis (PM). The susceptibility of mast cell-deficient WBB6F1-Kit^W/Kit^Wv mice (W/W^v mice) to a murine model of polymyositis, C protein-induced myositis (CIM), was compared with that of wild-type (WT) mice. The effect of mast cell reconstitution with bone marrow-derived mast cells (BMMCs) on the susceptibility of W/W^v mice to CIM was also evaluated.

Results

The number of mast cells in the affected muscle increased in patients with PM as compared with patients with DM. W/W^v mice exhibited significantly reduced disease incidence and histological scores of CIM as compared with WT mice. The number of CD8⁺ T cells and macrophages in the skeletal muscles of CIM decreased in W/W^v mice compared with WT mice. Engraftment of BMMCs restored the incidence and histological scores of CIM in W/W^v mice. Vascular permeability in the skeletal muscle was elevated in WT mice but not in W/W^v mice upon CIM induction.

Conclusion

Mast cells are involved in the pathogenesis of inflammatory myopathy.     

Activated MCTC mast cells infiltrate diseased lung areas in cystic fibrosis and idiopathic pulmonary fibrosis

Background

Although mast cells are regarded as important regulators of inflammation and tissue remodelling, their role in cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) has remained less studied. This study investigates the densities and phenotypes of mast cell populations in multiple lung compartments from patients with CF, IPF and never smoking controls.

Methods

Small airways, pulmonary vessels, and lung parenchyma were subjected to detailed immunohistochemical analyses using lungs from patients with CF (20 lung regions; 5 patients), IPF (21 regions; 7 patients) and controls (16 regions; 8 subjects). In each compartment the densities and distribution of MC_T and MC_TC mast cell populations were studied as well as the mast cell expression of IL-6 and TGF-β.

Results

In the alveolar parenchyma in lungs from patients with CF, MC_TC numbers increased in areas showing cellular inflammation or fibrosis compared to controls. Apart from an altered balance between MC_TC and MC_T cells, mast cell in CF lungs showed elevated expression of IL-6. In CF, a decrease in total mast cell numbers was observed in small airways and pulmonary vessels. In patients with IPF, a significantly elevated MC_TC density was present in fibrotic areas of the alveolar parenchyma with increased mast cell expression of TGF-β. The total mast cell density was unchanged in small airways and decreased in pulmonary vessels in IPF. Both the density, as well as the percentage, of MC_TC correlated positively with the degree of fibrosis. The increased density of MC_TC, as well as MC_TC expression of TGF-β, correlated negatively with patient lung function.

Conclusions

The present study reveals that altered mast cell populations, with increased numbers of MC_TC in diseased alveolar parenchyma, represents a significant component of the histopathology in CF and IPF. The mast cell alterations correlated to the degree of tissue remodelling and to lung function parameters. Further investigations of mast cells in these diseases may open for new therapeutic strategies.     

Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells

Background

Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Infections of the respiratory tract are a hallmark in CF. The host immune responses in CF are not adequate to eradicate pathogens, such as P. aeruginosa. Dendritic cells (DC) are crucial in initiation and regulation of immune responses. Changes in DC function could contribute to abnormal immune responses on multiple levels. The role of DC in CF lung disease remains unknown.

Methods

This study investigated the expression of CFTR gene in bone marrow-derived DC. We compared the differentiation and maturation profile of DC from CF and wild type (WT) mice. We analyzed the gene expression levels in DC from naive CF and WT mice or following P. aeruginosa infection.

Results

CFTR is expressed in DC with lower level compared to lung tissue. DC from CF mice showed a delayed in the early phase of differentiation. Gene expression analysis in DC generated from naive CF and WT mice revealed decreased expression of Caveolin-1 (Cav1), a membrane lipid raft protein, in the CF DC compared to WT DC. Consistently, protein and activity levels of the sterol regulatory element binding protein (SREBP), a negative regulator of Cav1 expression, were increased in CF DC. Following exposure to P. aeruginosa, expression of 3β-hydroxysterol-Δ7 reductase (Dhcr7) and stearoyl-CoA desaturase 2 (Scd2), two enzymes involved in the lipid metabolism that are also regulated by SREBP, was less decreased in the CF DC compared to WT DC.

Conclusion

These results suggest that CFTR dysfunction in DC affects factors involved in membrane structure and lipid-metabolism, which may contribute to the abnormal inflammatory and immune response characteristic of CF.     

Characterization of Nasal Potential Difference in cftr Knockout and F508del-CFTR Mice

Background

Treatments designed to correct cystic fibrosis transmembrane conductance regulator (CFTR) defects must first be evaluated in preclinical experiments in the mouse model of cystic fibrosis (CF). Mice nasal mucosa mimics the bioelectric defect seen in humans. The use of nasal potential difference (V_TE) to assess ionic transport is a powerful test evaluating the restoration of CFTR function. Nasal V_TE in CF mice must be well characterized for correct interpretation.

Methods

We performed V_TE measurements in large-scale studies of two mouse models of CF—B6;129 cftr knockout and FVB F508del-CFTR—and their respective wild-type (WT) littermates. We assessed the repeatability of the test for cftr knockout mice and defined cutoff points distinguishing between WT and F508del-CFTR mice.

Results

We determined the typical V_TE values for CF and WT mice and demonstrated the existence of residual CFTR activity in F508del-CFTR mice. We characterized intra-animal variability in B6;129 mice and defined the cutoff points for F508del-CFTR chloride secretion rescue. Hyperpolarization of more than -2.15 mV after perfusion with a low-concentration Cl^- solution was considered to indicate a normal response.

Conclusions

These data will make it possible to interpret changes in nasal V_TE in mouse models of CF, in future preclinical studies.     

MicroRNA-21 Knockout Improve the Survival Rate in DSS Induced Fatal Colitis through Protecting against Inflammation and Tissue Injury

Background

MicroRNA-21 (miR-21) is overexpressed in most inflammatory diseases, but its physiological role in gut inflammation and tissue injury is poorly understood. The goal of this work is to understand the role of miR-21 in colitis and damage progression of intestine in a genetically modified murine model.

Methods

Experimental colitis was induced in miR-21 KO and wild-type (WT) mice by 3.5% dextran sulphate sodium (DSS) administration for 7 days. Disease activity index(DAI), blood parameters, intestinal permeability, histopathologic injury, cytokine and chemokine production, and epithelial cells apoptosis were examined in colons of miR-21 KO and WT mice.

Results

miR-21 was overexpressed in intestine of inflammatory bowel diseases (IBD) and acute intestinal obstruction (AIO) patients when compared with normal intestinal tissues. Likewise, miR-21 was up-regulated in colon of IL-10 KO mice when compared with control mice. WT mice rapidly lost weight and were moribund 5 days after treatment with 3.5% DSS, while miR-21 KO mice survived for at least 6 days. Elevated leukocytes and more severe histopathology were observed in WT mice when compared with miR-21 KO mice. Elevated levels of TNF-α and macrophage inflammatory protein-2(MIP-2) in colon culture supernatants from WT mice exhibited significant higher than miR-21 KO mice. Furthermore, CD3 and CD68 positive cells, intestinal permeability and apoptosis of epithelial cells were significantly increased in WT mice when compared with miR-21 KO mice. Finally, we found that miR-21 regulated the intestinal barrier function through modulating the expression of RhoB and CDC42.

Conclusion

Our results suggest that miR-21 is overexpressed in intestinal inflammation and tissue injury, while knockout of miR-21 in mice improve the survival rate in DSS-induced fatal colitis through protecting against inflammation and tissue injury. Therefore, attenuated expression of miR-21 in gut may prevent the onset or progression of inflammatory bowel disease in patients.     

The Dual Effect of Cannabinoid Receptor-1 Deficiency on the Murine Postoperative Ileus

Introduction

Intestinal inflammatory responses play a critical role in the pathogenesis of postoperative ileus (POI). As cannabinoid receptor-1 (CB1) is involved in inhibiting gastrointestinal (GI) motility and anti-inflammation, we aimed to explore its contribution to POI.

Methods

Experimental POI was induced in adult female CB1-deficient (CB1–/–) mice and wild-type littermates (C57BL/6N) by standardized small bowel manipulation. Twenty-four hours after surgery, GI transit was assessed by charcoal transport. FITC avidin, F4/80, and myeloperoxidase immunohistochemistry techniques were used to evaluate the inflammatory response in the muscularis of ileum and colon. Expressions of p38MAPK and its phosphorylated form (pp38) in the intestine were determined. Plasma levels of proinflammatory cytokines and chemokines were measured by ELISA as well.

Results

POI was characterized by decreased GI transit (p<0.01) and accompanied by a marked intestinal and systematic inflammatory response in wild-type and CB1–/– mice. Increased numbers of inflammatory cells, including macrophages, neutrophils, and mast cells were observed in the muscularis of ileum and colon (p<0.01, or p<0.05). Plasma levels of interleukin-6 (IL-6), cytokine-induced neutrophil chemoattractant-1 (CINC-1/KC), and monocyte chemoattractant protein-1 (MCP-1) were elevated (p<0.01, or p<0.05). Expression of p38 and pp38 increased in the intestine (p<0.01, or p<0.05). CB1–/– mice showed an increased inflammatory response during POI, especially the systemic inflammatory markers, such as IL-6, KC, CINC1, and pp38 expression were increased as compared to those in WT mice (p<0.05).

Conclusions

Intestinal motility was inhibited during POI. In this condition, inhibition of motility did not seem to be altered by the absence of CB1 receptors, however, an increased inflammatory response was observed in CB1–/– mice. Hence, CB1 receptor activation rather than inhibition may reduce the inflammatory response in POI, which has a remote potential to relate into reduced inhibition of intestinal motility during POI.     

Guanylyl Cyclase-G Modulates Jejunal Apoptosis and Inflammation in Mice with Intestinal Ischemia and Reperfusion

Background

Membrane bound guanylyl cyclase-G (mGC-G), a novel form of GC mediates ischemia and reperfusion (IR)-induced renal injury. We investigated the roles of mGC-G in intestinal IR-induced jejunal damage, inflammation, and apoptosis.

Materials and methods

Male C57BL/6 wild-type (WT) and mGC-G gene knockout (KO) mice were treated with a sham operation or 45 min of superior mesenteric arterial obstruction followed by 3, 6, 12, or 24 h of reperfusion.

Results

Sham-operated KO mice had significantly lower plasma nitrate and nitrite (NOx) levels and jejunal villus height, crypt depth, and protein expression of phosphorylated-nuclear factor-kappa-B (NF-κB), phosphorylated-c-Jun N-terminal kinases (JNK) 2/3, phosphorylated-p38, and B-cell lymphoma-2 (Bcl-2). They had significantly greater jejunal interleukin-6 mRNA, cytochrome c protein, and apoptotic index compared with sham-operated WT mice. Intestinal IR significantly decreased plasma NOx in WT mice and increased plasma NOx in KO mice. The jejunal apoptotic index and caspase 3 activities were significantly increased, and nuclear phosphorylated-NF-κB and phosphorylated-p38 protein were significantly decreased in WT, but not KO mice with intestinal IR. After reperfusion, KO mice had an earlier decrease in jejunal cyclic GMP, and WT mice had an earlier increase in jejunal proliferation and a later increase in cytosol inhibitor of kappa-B-alpha. Intestinal IR induced greater increases in plasma and jejunal interleukin-6 protein in WT mice and a greater increase in jejunal interleukin-6 mRNA in KO mice.

Conclusions

mGC-G is involved in the maintenance of jejunal integrity and intestinal IR-induced inflammation and apoptosis. These results suggest that targeting cGMP pathway might be a potential strategy to alleviate IR-induced jejunal damages.     

Loss of guanylyl cyclase C (GCC) signaling leads to dysfunctional intestinal barrier

Background

Guanylyl Cyclase C (GCC) signaling via uroguanylin (UGN) and guanylin activation is a critical mediator of intestinal fluid homeostasis, intestinal cell proliferation/apoptosis, and tumorigenesis. As a mechanism for some of these effects, we hypothesized that GCC signaling mediates regulation of intestinal barrier function.

Methodology/Principal Findings

Paracellular permeability of intestinal segments was assessed in wild type (WT) and GCC deficient (GCC−/−) mice with and without lipopolysaccharide (LPS) challenge, as well as in UGN deficient (UGN−/−) mice. IFNγ and myosin light chain kinase (MLCK) levels were determined by real time PCR. Expression of tight junction proteins (TJPs), phosphorylation of myosin II regulatory light chain (MLC), and STAT1 activation were examined in intestinal epithelial cells (IECs) and intestinal mucosa. The permeability of Caco-2 and HT-29 IEC monolayers, grown on Transwell filters was determined in the absence and presence of GCC RNA interference (RNAi). We found that intestinal permeability was increased in GCC−/− and UGN−/− mice compared to WT, accompanied by increased IFNγ levels, MLCK and STAT1 activation in IECs. LPS challenge promotes greater IFNγ and STAT1 activation in IECs of GCC−/− mice compared to WT mice. Claudin-2 and JAM-A expression were reduced in GCC deficient intestine; the level of phosphorylated MLC in IECs was significantly increased in GCC−/− and UGN−/− mice compared to WT. GCC knockdown induced MLC phosphorylation, increased permeability in IEC monolayers under basal conditions, and enhanced TNFα and IFNγ-induced monolayer hyperpermeability.

Conclusions/Significance

GCC signaling plays a protective role in the integrity of the intestinal mucosal barrier by regulating MLCK activation and TJ disassembly. GCC signaling activation may therefore represent a novel mechanism in maintaining the small bowel barrier in response to injury.     

Role of Mast Cells in Inflammatory Bowel Disease and Inflammation-Associated Colorectal Neoplasia in IL-10-Deficient Mice

Background

Inflammatory bowel disease (IBD) is hypothesized to result from stimulation of immune responses against resident intestinal bacteria within a genetically susceptible host. Mast cells may play a critical role in IBD pathogenesis, since they are typically located just beneath the intestinal mucosal barrier and can be activated by bacterial antigens.

Methodology/Principal Findings

This study investigated effects of mast cells on inflammation and associated neoplasia in IBD-susceptible interleukin (IL)-10-deficient mice with and without mast cells. IL-10-deficient mast cells produced more pro-inflammatory cytokines in vitro both constitutively and when triggered, compared with wild type mast cells. However despite this enhanced in vitro response, mast cell-sufficient Il10 ^−/− mice actually had decreased cecal expression of tumor necrosis factor (TNF) and interferon (IFN)-γ mRNA, suggesting that mast cells regulate inflammation in vivo. Mast cell deficiency predisposed Il10 ^−/− mice to the development of spontaneous colitis and resulted in increased intestinal permeability in vivo that preceded the development of colon inflammation. However, mast cell deficiency did not affect the severity of IBD triggered by non-steroidal anti-inflammatory agents (NSAID) exposure or helicobacter infection that also affect intestinal permeability.

Conclusions/Significance

Mast cells thus appear to have a primarily protective role within the colonic microenvironment by enhancing the efficacy of the mucosal barrier. In addition, although mast cells were previously implicated in progression of sporadic colon cancers, mast cells did not affect the incidence or severity of colonic neoplasia in this inflammation-associated model.     

Increased plasma membrane cholesterol in cystic fibrosis cells correlates with CFTR genotype and depends on de novo cholesterol synthesis

Background

Previous observations demonstrate that Cftr-null cells and tissues exhibit alterations in cholesterol processing including perinuclear cholesterol accumulation, increased de novo synthesis, and an increase in plasma membrane cholesterol accessibility compared to wild type controls. The hypothesis of this study is that membrane cholesterol accessibility correlates with CFTR genotype and is in part influenced by de novo cholesterol synthesis.

Methods

Electrochemical detection of cholesterol at the plasma membrane is achieved with capillary microelectrodes with a modified platinum coil that accepts covalent attachment of cholesterol oxidase. Modified electrodes absent cholesterol oxidase serves as a baseline control. Cholesterol synthesis is determined by deuterium incorporation into lipids over time. Incorporation into cholesterol specifically is determined by mass spectrometry analysis. All mice used in the study are on a C57Bl/6 background and are between 6 and 8 weeks of age.

Results

Membrane cholesterol measurements are elevated in both R117H and ΔF508 mouse nasal epithelium compared to age-matched sibling wt controls demonstrating a genotype correlation to membrane cholesterol detection. Expression of wt CFTR in CF epithelial cells reverts membrane cholesterol to WT levels further demonstrating the impact of CFTR on these processes. In wt epithelial cell, the addition of the CFTR inhibitors, Gly H101 or CFTR_inh-172, for 24 h surprisingly results in an initial drop in membrane cholesterol measurement followed by a rebound at 72 h suggesting a feedback mechanism may be driving the increase in membrane cholesterol. De novo cholesterol synthesis contributes to membrane cholesterol accessibility.

Conclusions

The data in this study suggest that CFTR influences cholesterol trafficking to the plasma membrane, which when depleted, leads to an increase in de novo cholesterol synthesis to restore membrane content.     

Nucleotide-Oligomerization-Domain-2 Affects Commensal Gut Microbiota Composition and Intracerebral Immunopathology in Acute Toxoplasma gondii Induced Murine Ileitis

Background

Within one week following peroral high dose infection with Toxoplasma (T.) gondii, susceptible mice develop non-selflimiting acute ileitis due to an underlying Th1-type immunopathology. The role of the innate immune receptor nucleotide-oligomerization-domain-2 (NOD2) in mediating potential extra-intestinal inflammatory sequelae including the brain, however, has not been investigated so far.

Methodology/Principal Findings

Following peroral infection with 100 cysts of T. gondii strain ME49, NOD2^-/- mice displayed more severe ileitis and higher small intestinal parasitic loads as compared to wildtype (WT) mice. However, systemic (i.e. splenic) levels of pro-inflammatory cytokines such as TNF-α and IFN-γ were lower in NOD2^-/- mice versus WT controls at day 7 p.i. Given that the immunopathological outcome might be influenced by the intestinal microbiota composition, which is shaped by NOD2, we performed a quantitative survey of main intestinal bacterial groups by 16S rRNA analysis. Interestingly, Bifidobacteria were virtually absent in NOD2^-/- but not WT mice, whereas differences in remaining bacterial species were rather subtle. Interestingly, more distinct intestinal inflammation was accompanied by higher bacterial translocation rates to extra-intestinal tissue sites such as liver, spleen, and kidneys in T. gondii infected NOD2^-/- mice. Strikingly, intracerebral inflammatory foci could be observed as early as seven days following T. gondii infection irrespective of the genotype of animals, whereas NOD2^-/- mice exhibited higher intracerebral parasitic loads, higher F4/80 positive macrophage and microglia numbers as well as higher IFN-γ mRNA expression levels as compared to WT control animals.

Conclusion/Significance

NOD2 signaling is involved in protection of mice from T. gondii induced acute ileitis. The parasite-induced Th1-type immunopathology at intestinal as well as extra-intestinal sites including the brain is modulated in a NOD2-dependent manner.     

Mast cell-derived histamine mediates cystitis pain

Background

Mast cells trigger inflammation that is associated with local pain, but the mechanisms mediating pain are unclear. Interstitial cystitis (IC) is a bladder disease that causes debilitating pelvic pain of unknown origin and without consistent inflammation, but IC symptoms correlate with elevated bladder lamina propria mast cell counts. We hypothesized that mast cells mediate pelvic pain directly and examined pain behavior using a murine model that recapitulates key aspects of IC.

Methods and Findings

Infection of mice with pseudorabies virus (PRV) induces a neurogenic cystitis associated with lamina propria mast cell accumulation dependent upon tumor necrosis factor alpha (TNF), TNF-mediated bladder barrier dysfunction, and pelvic pain behavior, but the molecular basis for pelvic pain is unknown. In this study, both PRV-induced pelvic pain and bladder pathophysiology were abrogated in mast cell-deficient mice but were restored by reconstitution with wild type bone marrow. Pelvic pain developed normally in TNF- and TNF receptor-deficient mice, while bladder pathophysiology was abrogated. Conversely, genetic or pharmacologic disruption of histamine receptor H1R or H2R attenuated pelvic pain without altering pathophysiology.

Conclusions

These data demonstrate that mast cells promote cystitis pain and bladder pathophysiology through the separable actions of histamine and TNF, respectively. Therefore, pain is independent of pathology and inflammation, and histamine receptors represent direct therapeutic targets for pain in IC and other chronic pain conditions.     

Interleukin 13 and Serotonin: Linking the Immune and Endocrine Systems in Murine Models of Intestinal Inflammation

Objective

Infiltration of activated immune cells and increased cytokine production define the immunophenotype of gastrointestinal (GI) inflammation. In addition, intestinal inflammation is accompanied by alteration in the numbers of serotonin (5-hydroxytryptamine; 5-HT) synthesizing enterochromaffin (EC) cells and in 5-HT amount. It has been established that EC cells express interleukin (IL)-13 receptor, additionally IL-13 has been implicated in the pathogenesis of ulcerative colitis. In this study, we investigated the role of IL-13 mediated 5-HT signaling in pathogenesis of colitis.

Methodology

Colitis was induced in IL-13 deficient (IL-13−/−) and wild-type (WT) mice with dextran sulfate sodium (DSS) and dinitrobenzene sulfonic acid (DNBS), as well as in IL-13−/− mice given recombinant mouse IL-13 (rmIL-13) and 5-hydroxytryptamine (5-HTP), the direct precursor of 5-HT.

Principal Findings and Conclusion

Elevated colonic IL-13 levels were observed in WT mice receiving DSS in comparison to control. IL-13−/− mice administered DSS exhibited significantly reduced severity of colitis compared to WT mice as reflected by macroscopic and histological damage assessments. Following DSS administration, significantly lower pro-inflammatory cytokine production and fewer infiltrating macrophages were observed in IL-13−/− mice compared to WT. The reduced severity of colitis observed in IL-13−/− mice was also accompanied by down-regulation of EC cell numbers and colonic 5-HT content. In addition, increasing colonic 5-HT content by administration of rmIL-13 or 5-HTP exacerbated severity of DSS colitis in IL-13−/− mice. IL-13−/− mice also exhibited reduced severity of DNBS-induced colitis. These results demonstrate that IL-13 plays a critical role in the pathogenesis of experimental colitis and 5-HT is an important mediator of IL-13 driven intestinal inflammation. This study revealed important information on immune-endocrine axis in gut in relation to inflammation which may ultimately lead to better strategy in managing various intestinal inflammatory conditions including inflammatory bowel disease.     

Influence of language and ancestry on genetic structure of contiguous populations: A microsatellite based study on populations of Orissa

Background

Although cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the severity of disease is highly variable indicating the influence of modifier genes. The intestines of Cftr deficient mice (CF mice: Cftr ^tm1Unc ) are prone to obstruction by excessive mucus accumulation and are used as a model of meconium ileus and distal intestinal obstruction syndrome. This phenotype is strongly dependent on the genetic background of the mice. On the C57Bl/6 background, the majority of CF mice cannot survive on solid mouse chow, have inflammation of the small intestine, and are about 30% smaller than wild type littermates. In this work potential modifier loci of the CF intestinal phenotype were identified.

Results

CF mice on a mixed genetic background (95% C57Bl/6 and 5% 129Sv) were compared to CF mice congenic on the C57Bl/6 background for several parameters of the intestinal CF phenotype. CF mice on the mixed background exhibit significantly greater survival when fed dry mouse chow, have reduced intestinal inflammation as measured by quantitative RT-PCR for marker genes, have near normal body weight gain, and have reduced mucus accumulation in the intestinal crypts. There was an indication of a gender effect for body weight gain: males did not show a significant improvement at 4 weeks of age, but were of normal weight at 8 weeks, while females showed improvement at both 4 and 8 weeks. By a preliminary genome-wide PCR allele scanning, three regions were found to be potentially associated with the milder phenotype. One on chr.1, defined by marker D1Mit36, one on chr. 9 defined by marker D9Mit90, and one on chr. 10, defined by marker D10Mit14.

Conclusion

Potential modifier regions were found that have a positive impact on the inflammatory phenotype of the CF mouse small intestine and animal survival. Identification of polymorphisms in specific genes in these regions should provide important new information about genetic modifiers of the CF intestinal phenotype.     

Regulatory CD4+Foxp3+ T Cells Control the Severity of Anaphylaxis

Objective

Anaphylaxis is a life-threatening outcome of immediate-type hypersensitivity to allergen, consecutive to mast cell degranulation by allergen-specific IgE. Regulatory T cells (Treg) can control allergic sensitization and mast cell degranulation, yet their clinical benefit on anaphylactic symptoms is poorly documented. Here we investigated whether Treg action during the effector arm of the allergic response alleviates anaphylaxis.

Methods

We used a validated model of IgE-mediated passive systemic anaphylaxis, induced by intravenous challenge with DNP-HSA in mice passively sensitized with DNP-specific IgE. Anaphylaxis was monitored by the drop in body temperature as well as plasma histamine and serum mMCP1 levels. The role of Treg was analyzed using MHC class II-deficient (Aβ^°/°) mice, treatment with anti-CD25 or anti-CD4 mAbs and conditional ablation of Foxp3⁺ Treg in DEREG mice. Therapeutic efficacy of Treg was also evaluated by transfer experiments using FoxP3-eGFP knock-in mice.

Results

Anaphylaxis did not occur in mast cell-deficient W/W^v mutant mice and was only moderate and transient in mice deficient for histamine receptor-1. Defects in constitutive Treg, either genetic or induced by antibody or toxin treatment resulted in a more severe and/or sustained hypothermia, associated with a rise in serum mMCP1, but not histamine. Adoptive transfer of Foxp3⁺ Treg from either naïve or DNP-sensitized donors similarly alleviated body temperature loss in Treg-deficient DEREG mice.

Conclusion

Constitutive Foxp3⁺ Treg can control the symptomatic phase of mast cell and IgE-dependent anaphylaxis in mice. This might open up new therapeutic avenues using constitutive rather than Ag-specific Treg for inducing tolerance in allergic patients.