Development of low-dose streptozotocin-induced diabetes in ICAM-1-deficient mice.

Multiple injections of low-dose streptozotocin (LDSZ) induce immune-mediated insulitis and diabetes in C57BL/6 (H-2b) mice. To evaluate the role of the intercellular adhesion molecule-1 (ICAM-1) for LDSZ induced immune-mediated diabetes, we have investigated mice genetically deficient in the ICAM-1 gene (ICAM-1-/-) in comparison to wild-type (ICAM-1+/+) mice. ICAM-1-/- mice, which had a mixed genetic background of C57BL/6 and DBA/2 mice, were backcrossed to C57BL/6 mice and screened for H2b homogenicity. Mice received five daily injections of 40 mg/kg streptozotocin. On day 21 after the first LDSZ injection 55% of the ICAM-1+/+ (female 33%, male 80%) and 50% of the ICAM-1-/- (female 20%, male 100%), mice had blood glucose levels over 200 mg/dl. Mean blood glucose levels increased in response to LDSZ treatment, however, no differences between ICAM-1+/+ and ICAM-1-/- mice were noted. Histological examinations of pancreatic islets revealed mononuclear infiltration of pancreatic islets without significant differences between both groups of mice. In summary, LDSZ-induced immune-mediated insulitis and diabetes development occurs in ICAM-1-/- mice similarly than in ICAM-1+/+ mice. These results do not support the hypothesis that ICAM-1 plays a key role during immune-mediated infiltration and destruction of pancreatic islets in LDSZ induced diabetes.     

Knockout of Mkp-1 exacerbates colitis in Il-10-deficient mice

Il-10-deficient mice develop colitis associated with exaggerated Th1/Th17 responses and are a valuable model of inflammatory bowel disease. Mkp-1 is a major negative regulator of MAPKs, and its expression is enhanced by IL-10. To understand the role of Mkp-1 in the regulation of intestinal mucosal immune responses, we studied the effect of Mkp-1 deletion on the pathogenesis of colitis in Il-10(-/-) mice. We found that knockout of Mkp-1 on an Il-10(-/-) background accelerated the development of colitis. Compared with Il-10(-/-) mice, colitis not only appeared earlier but also was more severe in Il-10(-/-)/Mkp-1(-/-) mice. Il-10(-/-) mice exhibited a mild intestinal inflammation in the specific pathogen-free environment, and rectal prolapse rarely appeared before 6 mo of age. In contrast, the majority of Il-10(-/-)/Mkp-1(-/-) mice developed severe colitis rapidly and presented with rectal prolapse after only 2-3 mo. The colon of Il-10(-/-)/Mkp-1(-/-) mice showed diffuse transmural chronic inflammation and mucosal hyperplasia, with significantly more proliferating crypt epithelial cells than those of Il-10(-/-) mice. In addition to the severe colitis, Il-10(-/-)/Mkp-1(-/-) mice also developed conjunctivitis and blepharitis. The colon of Il-10(-/-)/Mkp-1(-/-) mice contained significantly higher levels of proinflammatory cytokines and exhibited greater MAPK activities than did the colon of Il-10(-/-) mice. Splenocytes and lymphocytes from Il-10(-/-)/Mkp-1(-/-) mice produced higher levels of Th1 cytokines ex vivo upon activation than did cells from Il-10(-/-) mice. Our studies support a pivotal role of Mkp-1 as a negative regulator of mucosal immune responses and highlight its protective function against inflammatory bowel disease.     

Beneficial effect of auto-aggregating Lactobacillus crispatus on experimentally induced colitis in mice

We tested the therapeutic relevance of auto aggregation in lactobacilli by comparing the effect on DSS induced colitis of viable Lactobacillus crispatus M247, isolated from healthy humans, to L. crispatus MU5, an isogenic spontaneous mutants of M247, the latter lacking the auto aggregation phenotype which allows the adhesion to human mucus. Aggregating L. crispatus M247, but not the non-aggregating MU5, was retrievable from mice feces and adherent to the colonic mucosa. Daily administration of L. crispatus M247, but not heat killed L. crispatus M247 or aggregation deficient L. crispatus MU5, dose-dependently reduced the severity of DSS colitis. Indeed, L. crispatus MU5 administered in a 30% sucrose solution, known to restore the aggregation phenotype, had a protective effect comparable to mice receiving L. crispatus M247. These results indicate that a surface-mediated property such as aggregation may play a pivotal role in the protective effects obtained by dietary supplementation with L. crispatus M247 during colitis.     

Treatment and mechanism of fecal microbiota transplantation in mice with experimentally induced ulcerative colitis

Restoring intestinal microbiota dysbiosis with fecal microbiota transplantation is considered as a promising treatment for ulcerative colitis. However, the mechanisms underlying its relieving effects remain unclear. Ulcerative colitis pathogenesis is associated with the involvement of immune cells and inflammatory cytokines. Here, we aimed to investigate the effect of fecal microbiota transplantation on T cell cytokines in a dextran sulfate sodium-induced ulcerative colitis mouse model. Five-aminosalicylic acid (5-ASA) was used as the positive control. Male C57BL/6 mice were randomly assigned to control, model (UC), UC + FMT, and UC + 5-ASA groups. Each group consisted of five mice. The establishment of the mouse model was verified by fecal occult-blood screening and hematoxylin–eosin staining. Results showed that fecal microbiota transplantation reduced colonic inflammation, significantly decreased T helper (Th)1 and Th17 cells, interferon-gamma, interleukin-2 and interleukin-17, as well as significantly increased Th2 and regulatory T (Treg) cells, interleukin-4, interleukin-10, and transforming growth factor-beta, and improved routine blood count. Furthermore, 16S rRNA gene-sequencing analysis showed a significant increase in the relative abundance of genus Akkermansia and a significant decrease in the relative abundance of genus Helicobacter in the ulcerative colitis group. Fecal microbiota transplantation restored the profile of the intestinal microbiota to that of the control group. These findings demonstrated the capability of fecal microbiota transplantation in controlling experimentally induced ulcerative colitis by improving Th1/Th2 and Th17/Treg imbalance through the regulation of intestinal microbiota.     

Histopathological and immunohistochemical study of the hepatic lesions experimentally induced by Entamoeba dispar

The sequence of hepatic necrotic-inflammatory events produced by Entamoeba dispar are originally described in this work. For the first time the experimental lesions produced by E. dispar were described in details, as well as the distribution of the trophozoites detected by the immunohistochemistry. Animals experimentally infected with E. dispar presented necrosis, thrombosis and chronic granulomatous inflammation. Immunoreactive products derived from trofozoites were observed close or associated with trophozoites, epithelioid cells, leucocytes and hepatocytes. Few are the articles on the literature about virulence of E. dispar, which is approximately 9 times more frequent than to Entamoeba histolytica (E. histolytica). Variation in the virulence is therefore expected and signalizing the need of the continuity of studies with E. dispar strains from different places in the world. Taking into account that E. dispar is a closely related species to E. histolytica, these studies could determine new elements involved with E. histolytica pathogenesis, helping us to better understand the disease.Key words: Entamoeba dispar, hepatic necrosis, granuloma, pathogenicity.     

Wallerian degeneration and axonal regeneration after sciatic nerve crush are altered in ICAM-1-deficient mice

The intercellular cell adhesion molecule-1 (ICAM-1) has been implicated in the recruitment of immune cells during inflammatory processes. Previous studies investigating its involvement in the process of Wallerian degeneration and focusing on its potential role in macrophage recruitement have come to controversial conclusions. To examine whether Wallerian degeneration is altered in the absence of ICAM-1, we have analyzed changes in the expression of axonal and Schwann cell markers following sciatic nerve crush in wildtype and ICAM-1-deficient mice. We report that the lack of ICAM-1 leads to impaired axonal degeneration and regeneration and to alterations in Schwann cell responses following sciatic nerve crush. Degradation of neurofilament protein, the collapse of axonal profiles, and the re-expression of neurofilament proteins are substantially delayed in the distal nerve segment of ICAM-1^-/- mice. In contrast, the degradation of myelin, as determined by immunostaining for myelin protein zero, is unaltered in the mutants. Upregulation of GAP-43 and p75 neurotrophin receptor (p75^NTR) expression, characteristic for Schwann cells dedifferentiating in response to nerve injury, is differentially altered in the mutant animals. These results indicate that ICAM-1 is essential for the normal progression of axonal degeneration and regeneration in distal segments of injured peripheral nerves.     

Leukocyte recruitment and neuroglial activation during facial nerve regeneration in ICAM-1-deficient mice: effects of breeding strategy

Intercellular adhesion molecule 1 (ICAM-1) is a widely expressed glycoprotein involved in leukocyte extravasation and the interaction of lymphocytes with antigen-presenting cells. We examined these aspects of ICAM-1 function in the central nervous system after axonal injury in wild-type and ICAM-1-deficient mice. ICAM-1 immunoreactivity in the normal mouse facial nucleus was restricted to the vascular endothelium. Transection of the facial nerve led to a fast upregulation of ICAM-1 on activated microglia in the axotomized facial nucleus and the infiltration of ICAM-1-positive lymphocytes. Labeling elsewhere was unchanged. In homozygous ICAM-1 mutant mice, ICAM-1 was absent from endothelial cells and lymphocytes, but low levels of ICAM-1 were detected on cell membranes of reactive microglial cells. Comparison of wild-type animals with homozygously bred, ICAM-1-deficient mice showed a reduction of astrocytic and microglial activation, massive late axonal sprouting, and decreased lymphocyte infiltration. These experiments were repeated in F1 progeny of heterozygous mice on a C57BL/6 background. Neuroglial activation and lymphocyte infiltration in F1 homozygously deficient mice was unaffected compared with wild-type siblings. The invading ICAM-1-deficient lymphocytes also adhered to the ICAM-1-positive phagocytotic microglial cells in the ICAM-1 mutants. No change in the recruitment of macrophages and granulocytes into the crushed facial nerve, and no effect on axonal regeneration occurred. These data argue against the requirement of endothelial ICAM-1 in the recruitment of leukocytes into the crushed peripheral nerve or the axotomized facial motor nucleus and stress the importance of adequately matched controls in studying the effects of gene deletion in experimental animals.     

Diet-induced obesity and hepatic gene expression alterations in C57BL/6J and ICAM-1-deficient mice

The effects of high-fat feeding on the development of obesity were evaluated in intercellular adhesion molecule-1 (ICAM-1) knockout and C57BL/6J (B6) male mice fed a high-fat diet for < or =50 days. Serum and tissues were collected at baseline and after 1, 11, and 50 days on the diet. After 11 days on the diet, ICAM-1-deficient, but not B6, mice developed fatty livers and showed a significant increase in inguinal fat pad weight. At day 50, ICAM-1-deficient mice weighed less, and their adiposity index and circulating leptin levels were significantly lower than those of B6 controls. To better understand the early differential response to the diet, liver gene expression was analyzed at three time points by use of Affymetrix GeneChips. In both strains, a similar pattern of gene expression was detected in response to the high-fat diet. However, sterol regulatory element-binding protein-1, apolipoprotein A4, and adipsin mRNAs were significantly induced in ICAM-1-deficient livers, suggesting that these genes and their associated pathways may be involved in the acute diet response observed in the knockout mice.     

Osteoporosis in MCHR1-deficient mice

It is well recognized that the hypothalamus is of central importance in the regulation of food intake and fat mass. Recent studies indicate that it also plays an important role in the regulation of bone mass. Melanin concentrating hormone (MCH) is highly expressed in the hypothalamus and has been implicated in regulation of energy homeostasis. We developed MCHR1 inactivated mice to evaluate the physiological role of this receptor. Interestingly, the MCHR1(-/-) mice have osteoporosis, caused by a reduction in the cortical bone mass, while the amount of trabecular bone is unaffected. The reduction in cortical bone mass is due to decreased cortical thickness. Serum levels of c-telopeptide, a marker of bone resorption, are increased in MCHR1(-/-) mice, indicating that the MCHR1(-/-) mice have a high bone turnover osteoporosis. In conclusion, the MCHR1(-/-) mice have osteoporosis, indicating that MCHR1-signalling is involved in a tonic stimulation of bone mass.     

A model of Salmonella colitis with features of diarrhea in SLC11A1 wild-type mice

BACKGROUND: Mice do not get diarrhea when orally infected with S. enterica, but pre-treatment with oral aminoglycosides makes them susceptible to Salmonella colitis. However, genetically susceptible ItyS mice (Nramp1(G169D) allele) die from systemic infection before they develop diarrhea, so a new model is needed to study the pathogenesis of diarrhea. We pretreated ItyR mice (Nramp1(G169)) with oral kanamycin prior to infecting them with virulent S. Typhimurium strain 14028s in order to study Salmonella-induced diarrhea. We used both a visual scoring system and the measurement of fecal water content to measure diarrhea. BALB/c.D2(Nramp1) congenic started losing weight 5 days post-infection and they began to die from colitis 10-14 days after infection. A SPI-1 (invA) mutant caused cecal, but not colonic inflammation and did not cause diarrhea. A phoP- mutant did not cause manifestations of diarrhea in either normal or NADPH-deficient (gp91(phox)) mice. However, strain 14028s caused severe colitis and diarrhea in gp91(phox)-deficient mice on an ItyR background. pmr A and F mutants, which are less virulent in orally infected BALB/c mice, were fully virulent in this model of colitis. CONCLUSIONS: S. enterica must be able to invade the colonic epithelium and to persist in the colon in order to cause colitis with manifestations of diarrhea. The NADPH oxidase is not required for diarrhea in Salmonella colitis. Furthermore, a Salmonella phoP mutant can be cleared from the colon by non-oxidative host defenses.     

Identification of experimentally induced colitis by in vitro nuclear magnetic resonance

The present study determined whether in vitro nuclear magnetic resonance could be used to assess experimentally induced colitis in rats. Acute colitis was induced in 6 Sprague-Dawley rats by acetic acid enema, while 6 control animals received saline enemas. All animals were sacrificed 24 hours post-enema, and NMR relaxation times, T1 and T2, of colonic samples were determined on a 10 MHz spin analyzer (RADX, Houston, TX). Colonic water content was determined on the same samples by desiccation. Colitis animals showed significantly higher T1 and T2 relaxation times and tissue water content than controls. T1 and T2 times correlated significantly with tissue water content. Twelve additional animals were studied histologically, six of which received acetic acid enemas and showed extensive transmural colitis. Our results suggest that in vivo proton NMR might be a useful means of non-invasively assessing the degree of colonic inflammation.     

NHE3 modulates the severity of colitis in IL-10-deficient mice

NHE3, the major intestinal Na(+)/H(+) exchanger, was shown to be downregulated and/or inhibited in patients with inflammatory bowel disease (IBD), a phenomenon believed to contribute to inflammation-associated diarrhea. NHE3(-/-) mice spontaneously develop colitis and demonstrate high susceptibility to dextran sulfate-induced mucosal injury. We investigated the effects of NHE3 deficiency on the development of chronic colitis in an IL-10 knockout (KO) mouse model of Crohn's disease. NHE3(-/-) mice were first backcrossed to 129/SvEv mice for >10 generations, with no apparent changes in their survival or phenotype. These mice were crossed with IL-10(-/-) mice on the same genetic background, and the phenotypes of 10-wk-old wild-type (WT), IL-10(-/-), NHE3(-/-), and IL-10(-/-)/NHE3(-/-) (double-KO) mice were studied. Histological and immunohistochemical examination of the colon established important architectural alterations, including increased neutrophilic and mononuclear cell infiltration in double- compared with single-KO mice. Double-KO mice demonstrated increased colonic expression of neutrophil collagenase matrix metalloproteinase-8 and the chemokines macrophage inflammatory protein-2, CXCL1, CXCL10, and CXCL11. Colonic IFNγ, IL-17, and IL-12/23 p40 protein secretion was significantly increased in double- compared with single-KO mice. IL-10(-/-)/NHE3(-/-) mouse colonic epithelium exhibited increased hallmarks of apoptosis, including a significantly increased number of cleaved caspase-3-positive surface epithelial cells. These results highlight the importance of NHE3 in the maintenance of intestinal barrier integrity and in modulating the inflammatory process in IL-10-deficient mice. Chronic NHE3 inhibition or underexpression observed in IBD may therefore contribute to the pathogenesis of IBD by influencing the extent of the epithelial barrier defect and affect the ultimate degree of inflammation.     

Altered myogenesis in Six1-deficient mice

Six homeoproteins are expressed in several tissues, including muscle, during vertebrate embryogenesis, suggesting that they may be involved in diverse differentiation processes. To determine the functions of the Six1 gene during myogenesis, we constructed Six1-deficient mice by replacing its first exon with the lacZ gene. Mice lacking Six1 die at birth because of severe rib malformations and show extensive muscle hypoplasia affecting most of the body muscles in particular certain hypaxial muscles. Six1(-/-) embryos have impaired primary myogenesis, characterized, at E13.5, by a severe reduction and disorganisation of primary myofibers in most body muscles. While Myf5, MyoD and myogenin are correctly expressed in the somitic compartment in early Six1(-/-) embryos, by E11.5 MyoD and myogenin gene activation is reduced and delayed in limb buds. However, this is not the consequence of a reduced ability of myogenic precursor cells to migrate into the limb buds or of an abnormal apoptosis of myoblasts lacking Six1. It appears therefore that Six1 plays a specific role in hypaxial muscle differentiation, distinct from those of other hypaxial determinants such as Pax3, cMet, Lbx1 or Mox2.     

Colonic gene expression profile in NHE3-deficient mice: evidence for spontaneous distal colitis

Na+/H+ exchanger 3 (NHE3) provides a major route for intestinal Na+ absorption. NHE3 has been considered a target of proinflammatory cytokines and enteropathogenic bacteria, and impaired NHE3 expression and/or activity may be responsible for inflammation-associated diarrhea. However, the possibility of loss of NHE3 function reciprocally affecting gut immune homeostasis has not been investigated. In this report, we describe that NHE3-deficient mice spontaneously develop colitis restricted to distal colonic mucosa. NHE3(-/-) mice housed in a conventional facility exhibited phenotypic features such as mild diarrhea, occasional rectal prolapse, and reduced body weight. Genomewide microarray analysis identified not only a large group of transport genes that potentially represent an adaptive response, but also a considerable number of genes consistent with an inflammatory response. Histological examination demonstrated changes in the distal colon consistent with active inflammation, including crypt hyperplasia with an increased number of 5-bromo-2'-deoxyuridine-positive cells, diffuse neutrophilic infiltrate with concomitant 15-fold increase in matrix metalloproteinase 8 expression, an increased number of pSer276-RelA-positive cells, and a significant decrease in periodic acid-Schiff-positive goblet cells. Real-time PCR demonstrated elevated expression of inducible nitric oxide synthase (38-fold), TNF-alpha (6-fold), macrophage inflammatory protein-2 (48-fold), and IL-18 (3-fold) in the distal colon of NHE3(-/-) mice. NHE3(-/-) mice showed enhanced bacterial adhesion and translocation in the distal colon. Colitis was ameliorated by oral administration of broad-spectrum antibiotics. In conclusion, NHE3 deficiency leads to an exacerbated innate immune response, an observation suggesting a potentially novel role of NHE3 as a modifier gene, which when downregulated during infectious or chronic colitis may modulate the extent and severity of colonic inflammation.     

Enhanced pro-inflammatory cytokine production in Galphai2-deficient mice on colitis prone and colitis resistant 129Sv genetic backgrounds

Mice deficient in G-protein subunit alphai2 develop colitis closely resembling human ulcerative colitis when raised on 129SvEv background. When backcrossing the Galphai2-deficiency into a 129SvJBom genetic background, surprisingly, mice did not develop colitis. In vitro stimulation of splenocytes with formalin-killed Staphylococcus aureus resulted in significantly increased production of interleukin-1beta, tumor necrosis factor, and interleukin-12p40 in Galphai2(-/-) as compared to control mice. The enhanced production of pro-inflammatory cytokines was seen in colitis prone as well as in colitis resistant genetic background. A similar outcome was seen upon stimulation with toxic shock syndrome toxin-1, a T cell superantigen, except that Galphai2(-/-) colitis resistant 129SvJBom splenocytes did not show increased production of IL-12p40 as compared to their controls.     

Spermatogonial depletion in adult Pin1-deficient mice

Spermatogonia in the mouse testis arise from early postnatal gonocytes that are derived from primordial germ cells (PGCs) during embryonic development. The proliferation, self-renewal, and differentiation of spermatogonial stem cells provide the basis for the continuing integrity of spermatogenesis. We previously reported that Pin1-deficient embryos had a profoundly reduced number of PGCs and that Pin1 was critical to ensure appropriate proliferation of PGCs. The current investigation aimed to elucidate the function of Pin1 in postnatal germ cell development by analyzing spermatogenesis in adult Pin1-/- mice. Although Pin1 was ubiquitously expressed in the adult testis, we found it to be most highly expressed in spermatogonia and Sertoli cells. Correspondingly, we show here that Pin1 plays an essential role in maintaining spermatogonia in the adult testis. Germ cells in postnatal Pin1-/- testis were able to initiate and complete spermatogenesis, culminated by production of mature spermatozoa. However, there was a progressive and age-dependent degeneration of the spermatogenic cells in Pin1-/- testis that led to complete germ cell loss by 14 mo of age. This depletion of germ cells was not due to increased cell apoptosis. Rather, detailed analysis of the seminiferous tubules using a germ cell-specific marker revealed that depletion of spermatogonia was the first step in the degenerative process and led to disruption of spermatogenesis, which resulted in eventual tubule degeneration. These results reveal that the presence of Pin1 is required to regulate proliferation and/or cell fate of undifferentiated spermatogonia in the adult mouse testis.     

Defective osteoblast function in ICAP-1-deficient mice

The integrin receptor family plays important roles in cell-to-cell and cell-to-extracellular matrix interactions through the recruitment of accessory molecules. One of them, the integrin cytoplasmic domain-associated protein-1 (ICAP-1; also known as ITGB1BP1), specifically interacts with the cytoplasmic domain of the beta1 integrin subunit and negatively regulates its function in vitro. To address the role of ICAP-1 in vivo, we ablated the Icap-1 gene in mice. We report an unexpected role of ICAP-1 in osteoblast function during bone development. Icap-1-deficient mice suffer from reduced osteoblast proliferation and delayed bone mineralization, resulting in the retarded formation of bone sutures. In vitro studies reveal that primary and immortalized Icap-1-null osteoblasts display enhanced adhesion and spreading on extracellular matrix substrates, probably owing to an increase in beta1 integrin activation. Finally, we provide evidence that ICAP-1 promotes differentiation of osteoprogenitors by supporting their condensation through modulating the integrin high affinity state.     

Decreased blood pressure in NOX1-deficient mice

To understand the role of the superoxide-generating NADPH oxidase NOX1 in the vascular system, we have generated NOX1-deficient mice. NOX1-deficient mice had a moderately decreased basal blood pressure. In response to angiotensin II they showed an almost complete loss of the sustained blood pressure response, while the initial increase was conserved. NOX1-deficient mice showed a marked reduction in aortic media hypertrophy. Angiotensin II-induced smooth muscle cell proliferation was conserved, but there was a marked decrease in extracellular matrix accumulation. Our results establish a role for NOX1 in blood pressure regulation and vascular angiotensin II response.