Christine V. Whiting

The Role of Up-Regulated Serine Proteases and Matrix Metalloproteinases in the Pathogenesis of a Murine Model of Colitis

Proteinases are important at several phases of physiological and pathological inflammation, mediating cellular infiltration, cytokine activation, tissue damage, remodeling, and repair. However, little is known of their role in the pathogenesis of inflammatory bowel disease. The aim of this study was to assess the role of tissue proteases in a mouse model of colitis. Proteolytic activity was analyzed, using gel and in situ zymography, in colonic tissues from severe combined immunodeficient mice with colitis induced by transfer of CD4(+) T lymphocytes. Serine proteinase levels increased in colitic tissue, with major species of 23 kd, 30 kd, and 45 kd. Co-migration and inhibition studies indicated that the 23-kd proteinase was pancreatic trypsin and that the 30-kd species was neutrophil elastase. Matrix metalloproteinase (MMP)-9 expression, and MMP-2 and MMP-9 activation, was elevated in colitic tissues. Proteinase levels followed a decreasing concentration gradient from proximal to distal colon. Proteolysis was localized to infiltrating leukocytes in diseased severe combined immunodeficient mice. Transmural inflammation was associated with serine proteinase and MMP activity in overlying epithelium and with marked subepithelial proteolytic activity. The results demonstrate a clear elevation in the levels and activation of proteases in colitis, potentially contributing to disease progression through loss of epithelial barrier function.

Dietary n‐3 polyunsaturated fatty acids reduce disease and colonic proinflammatory cytokines in a mouse model of colitis

Background: n‐3 polyunsaturated fatty acids (PUFAs) reduce the severity of chronic inflammatory bowel disease, probably by means of reduction of immune cell activation or enhancement of the epithelial barrier. Using the severe combined immunodeficient (SCID) mouse model of colitis, this study examined the effect of dietary n‐3 PUFAs on development of colitis and on immunologic, epithelial, and matrix parameters in the intestines of control and colitic animals. Methods: SCID mice were fed n‐3‐enriched or control diet for 3 weeks before colitis induction by transplantation of CD45RBhigh T cells and maintained on the same diet for 4 to 8 weeks. Phenotype of infiltrating cells, epithelial ZO‐1 protein, and mucosal type I collagen were assessed by immunohistology and tissue cytokines by ELISA. Results: Transplanted n‐3‐fed animals had significantly reduced pathology scores, colonic tumor necrosis factor‐&agr;, interleukin‐12, and interleukin‐1&bgr; compared with animals fed standard diet. Proinflammatory cytokines were reduced despite a similar level of immune cell infiltration by T cells, CD11c+ cells, and CD11b+ cells. Neutrophil infiltration was significantly reduced in n‐3‐fed control and colitic mice, and other myeloid populations were reduced in mice on the n‐3 diet. Epithelial ZO‐1 expression was increased, and myofibroblast activation significantly decreased in transplanted n‐3‐fed animals compared with standard diet mice. Submucosal collagen synthesis was enhanced in n‐3‐fed mice. Conclusions: Dietary n‐3 PUFAs reduced clinical colitis and colonic immunopathology in this model of colonic inflammation by decreasing proinflammatory cytokine synthesis, reducing myeloid cell recruitment and activation, and enhancing epithelial barrier function and mucosal wound healing mechanisms.

IL-13 promotes collagen accumulation in Crohn's disease fibrosis by down-regulation of fibroblast MMP synthesis: a role for innate lymphoid cells?

Background Fibrosis is a serious consequence of Crohn’s disease (CD), often necessitating surgical resection. We examined the hypothesis that IL-13 may promote collagen accumulation within the CD muscle microenvironment. Methods Factors potentially modulating collagen deposition were examined in intestinal tissue samples from fibrotic (f) CD and compared with cancer control (C), ulcerative colitis (UC) and uninvolved (u) CD. Mechanisms attributable to IL-13 were analysed using cell lines derived from uninvolved muscle tissue and tissue explants. Results In fCD muscle extracts, collagen synthesis was significantly increased compared to other groups, but MMP-2 was not co-ordinately increased. IL-13 transcripts were highest in fCD muscle compared to muscle from other groups. IL-13 receptor (R) α1 was expressed by intestinal muscle smooth muscle, nerve and KIR+ cells. Fibroblasts from intestinal muscle expressed Rα1, phosphorylated STAT6 in response to IL-13, and subsequently down-regulated MMP-2 and TNF-α-induced MMP-1 and MMP-9 synthesis. Cells with the phenotype KIR+CD45+CD56+/−CD3− were significantly increased in fCD muscle compared to all other groups, expressed Rα1 and membrane IL-13, and transcribed high levels of IL-13. In explanted CD muscle, these cells did not phosphorylate STAT6 in response to exogenous IL-13. Conclusions The data indicate that in fibrotic intestinal muscle of Crohn’s patients, the IL-13 pathway is stimulated, involving a novel population of infiltrating IL-13Rα1+, KIR+ innate lymphoid cells, producing IL-13 which inhibits fibroblast MMP synthesis. Consequently, matrix degradation is down-regulated and this leads to excessive collagen deposition.

Vaginal and rectal infection of cats with feline immunodeficiency virus.

The objective of this study was to examine the potential of vaginal and rectal mucosal routes for feline immunodeficiency virus (FIV) uptake and infection, as a model of mucosal HIV infection, and to determine the fate of virus at these mucosal sites following transmission of infection. SPF cats were exposed to FIV isolates (PET, GL-8, T637), administered as either cell-associated or cell-free inocula, via the rectum or vagina. Establishment of infection was confirmed by isolation of infectious FIV from peripheral blood mononuclear cells (PBMC), and by presence of FIV proviral DNA in PBMC using a nested polymerase chain reaction. Fate of virus in tissue taken at necropsy from cats infected for 6-48 weeks was assessed by localizing FIV core and envelope proteins, p24 and gp41, using a biotin-streptavidin linked immunoperoxidase (IP) technique. Cells susceptible to infection were identified by an in situ hybridization technique for FIV viral DNA and RNA. Cell-free, as well as cell-associated, virus was infectious across intact vaginal and rectal mucosal surfaces. Transmission was most successful using cell-associated inocula, and via the rectal route. Cells infected with FIV were detected by IP staining in the colon of 6/9 rectally challenged cats and 1/5 vaginally challenged cats. Virus was predominantly localized within the epithelium at the base of the colonic crypts associated with lymphoid aggregates (follicle associated epithelium; FAE), and within the lymphoid follicle itself. Occasional infected cells were also noted within the lamina propria. The distribution of FIV DNA positive cells in the colon was similar to that for FIV antigen whilst FIV RNA positive cells were found more extensively, including within the lamina propria and lymphoid follicle. FIV infected cells were not detected within the vagina, or colonic and ileac lymph nodes. Similar patterns of infected cells were seen in all of the positive cats, indicating that colonic tissues remain persistently actively infected with FIV. We conclude that the FIV/cat model of rectal and vaginal mucosal infection should prove useful for characterizing the mechanism by which HIV infects mucosal surfaces and as a challenge system for the design of vaccines effective at preventing HIV infection via rectal and vaginal routes.

Vaccination with fixed feline immunodeficiency virus (FIV) infected cells: protection, breakthrough and specificity of response.

Infection of cats with feline immunodeficiency virus (FIV), a naturally occurring lentivirus infection of cats which causes an AIDS-like disease, has generated considerable interest as an animal model for HIV vaccination. This paper reports on experiments performed to examine the potential of a fixed infected cell vaccine to confer protection against intraperitoneal challenge with cell-free FIV. The cell vaccine was highly immunogenic and elicited antibody responses to virus core antigen, p24, high virus neutralizing (VN) antibody titres, and antibodies which recognized cellular components of the vaccine. Whilst protection, assessed by the inability to detect infectious virus by virus isolation or polymerase chain reaction, against homologous but not heterologous FIV isolates was apparent up to week 12 post-challenge, when cats were monitored longer up to week 50 post-challenge a breakthrough in vaccine protection against homologous virus was observed. Protection could not be correlated with levels of antibody to p24 or VN antibody titres. In contrast with simian immunodeficiency virus vaccine studies in macaques there was no clear evidence that antibodies recognizing cellular components of the vaccine, including MHC class I and II antigens, conferred any protective effect following challenge. These results indicate that long-term post-challenge monitoring for infection is essential in lentivirus vaccine trials.

Mucosal immunization with experimental feline immunodeficiency virus (FIV) vaccines induces both antibody and T cell responses but does not protect against rectal FIV challenge.

Feline immunodeficiency virus (FIV) is a natural lentiviral pathogen of cats which can be experimentally transmitted via rectal and vaginal routes--the major routes of human immunodeficiency virus type 1 transmission in man. An important objective for lentiviral research is the development of vaccine strategies which generate good mucosal immune responses capable of giving protection from a mucosal virus challenge. The experimental vaccines employed in this study were based on (a) a peptide from the third variable region of the FIV envelope glycoprotein and (b) fixed whole FIV, Glasgow-8 strain. Adjuvants used were Quil A and cholera toxin for mucosal administration and incomplete Freunds adjuvant and immune stimulating complexes for subcutaneous injection. Mucosal immunization was given by rectal and intranasal routes. Both antibody and proliferative responses were elicited by mucosal immunization and cholera toxin was found to be a good mucosal adjuvant. The addition of a lipo thioester to the FIV peptide improved IgG and IgA responses upon parenteral administration. However, no protection from a rectal FIV challenge was achieved.

Proinflammatory cytokine synthesis by mucosal fibroblasts from mouse colitis is enhanced by interferon-γ-mediated up-regulation of CD40 signalling

Gut mesenchymal fibroblasts form complex phenotypical and functional populations. They participate actively in homeostatic maintenance of the extracellular matrix, epithelial barrier function, repair mechanisms and leucocyte migration. In inflammation, they become activated, change matrix expression and synthesize proinflammatory mediators. Subpopulations of mucosal fibroblasts express CD40 and the aim of this study was to define its role in their proinflammatory function. Stable primary fibroblast lines derived from normal mouse colon and inflamed colon from CD4+ CD45RBhigh‐transplanted SCID mice were used as models to explore the role of mucosal fibroblast CD40 in the inflammatory process. Phenotype correlated with in situ fibroblast phenotype in the tissues of origin. Lines from both sources co‐expressed CD40 and Thy1·2 independently of α‐smooth muscle actin. A subpopulation of CD40+ fibroblasts from normal colon expressed CD40 at high levels and expression was enhanced by interferon (IFN)‐γ treatment, whereas all CD40+ fibroblasts from colitis expressed at low levels and expression was unaffected by IFN‐γ treatment. Despite lower‐level expression of CD40 by cells from colitis, they secreted constitutively interleukin (IL)‐6 and C‐C chemokine (CCL)2. Ligation of CD40 enhanced secretion of these mediators and induced secretion of CCL3. CD40 in cells from colitis was more responsive to ligation than CD40 on cells from normal tissue and this sensitivity was amplified selectively by the action of IFN‐γ. We conclude that the inflammatory milieu in colitis induces long‐lasting changes in phenotype and proinflammatory function in colonic fibroblasts. In particular, proinflammatory signalling from fibroblast CD40 is amplified synergistically by the Th1 effector T cell cytokine, IFN‐γ.

Abnormal Mucosal Extracellular Matrix Deposition Is Associated with Increased TGF-β Receptor-expressing Mesenchymal Cells in a Mouse Model of Colitis

Transforming growth factor-β (TGF-β) depresses mucosal inflammation and upregulates extracellular matrix (ECM) deposition. We analyzed TGF-β receptors RI and RII as well as ECM components using the CD4+ T-cell-transplanted SCID mouse model of colitis. The principal change in colitis was an increased proportion of TGF-β RII+ mucosal mesenchymal cells, predominantly α-smooth muscle actin (SMA)+ myofibroblasts, co-expressing vimentin and basement membrane proteins, but not type I collagen. TGF-β RII+ SMA− fibroblasts producing type I collagen were also increased, particularly in areas of infiltration and in ulcers. Type IV collagen and laminin were distributed throughout the gut lamina propria in disease but were restricted to the basement membrane in controls. In areas of severe epithelial damage, type IV collagen was lost and increased type I collagen was observed. To examine ECM production by these cells, mucosal mesenchymal cells were isolated. Cultured cells exhibited a similar phenotype and matrix profile to those of in vivo cells. The data suggested that there were at least two populations of mesenchymal cells responsible for ECM synthesis in the mucosa and that ligation of TGF-β receptors on these cells resulted in the disordered and increased ECM production observed in colitic mucosa.

Tumour necrosis factor-alpha (TNF-α) transcription and translation in the CD4+ T cell-transplanted scid mouse model of colitis

The adoptive transfer of activated CD4+α/β T cell blasts from the spleens of immunocompetent C.B‐17+/+ or BALB/cdm2 mice into C.B‐17scid/scid (scid) mice induces a colitis in the scid recipient within 8 weeks, which progresses to severe disease within 16 weeks. T cells isolated from recipient colon show a Th1 cytokine phenotype. We have examined the relationship between the phenotype of the cellular infiltrate and the transcription and translation of the proinflammatory cytokine TNF‐α. The techniques of double indirect immunohistology and in situ hybridization using digoxigenin‐labelled riboprobes were used. The prominent myeloid cell infiltrate in diseased tissues comprised F4/80+, Mac‐l+ macrophages, neutrophils, dendritic cells and activated macrophages. TNF‐α transcription and translation were associated with activated macrophages in the lamina propria. Activated macrophages transcribing and translating TNF‐α were clustered in areas of tissue destruction. Crypt epithelium of inflamed tissues transcribed TNF‐α at a very early stage of the disease process, but translation of TNF‐α protein could only be found in advanced epithelial dysplasia. This indicates differential post‐transcriptional control of TNF‐α in activated macrophages and the epithelium.

Stress-related changes to immune cells in the skin prior to wounding may impair subsequent healing

Higher psychological stress is associated with slower dermal wound healing, but the immunological mechanisms behind this effect are only partially understood. This paper aims to investigate whether immune cells present in the skin prior to wounding can affect subsequent healing in high-stress and low-stress participants. Two studies are presented in which skin biopsies were analysed using immunohistochemistry for numbers of macrophages and Langerhans cells, and immune cell activation (Study 2 only). Immune cells were related to perceived stress levels and subsequent healing. Study 1 included 19 healthy older adults and showed that higher stress was associated with significantly fewer macrophages in the skin. Study 2 included 22 younger adults and showed that higher stress was associated with significantly lower activation of immune cells in the skin. Furthermore, lower activation of immune cells (as measured by human leukocyte antigen (HLA expression)) and fewer Langerhans cells were associated with slower healing. Together these studies show the first preliminary evidence that the number and activation of immune cells in the skin prior to wounding are affected by stress and can impact healing. Larger studies are needed to confirm these effects.