Raymond A. Isidro

Colonic macrophage polarization in homeostasis, inflammation, and cancer.

Our review focuses on the colonic macrophage, a monocyte-derived, tissue-resident macrophage, and the role it plays in health and disease, specifically in inflammatory conditions such as inflammatory bowel disease and cancer of the colon and rectum. We give special emphasis to macrophage polarization, or phenotype, in these different states. We focus on macrophages because they are one of the most numerous leukocytes in the colon, and because they normally contribute to homeostasis through an anti-inflammatory phenotype. However, in conditions such as inflammatory bowel disease, proinflammatory macrophages are increased in the colon and have been linked to disease severity and progression. In colorectal cancer, tumor cells may employ anti-inflammatory macrophages to promote tumor growth and dissemination, whereas proinflammatory macrophages may antagonize tumor growth. Given the key roles that this cell type plays in homeostasis, inflammation, and cancer, the colonic macrophage is an intriguing therapeutic target. As such, potential macrophage-targeting strategies are discussed.

Serum vitamin D and colonic vitamin D receptor in inflammatory bowel disease

AIM To determine serum vitamin D levels and colonic vitamin D receptor (VDR) expression in inflammatory bowel disease (IBD) and non-IBD patients and correlate these with histopathology. METHODS Puerto Rican IBD (n = 10) and non-IBD (n = 10) patients ≥ 21 years old scheduled for colonoscopy were recruited. Each patient completed a questionnaire and provided a serum sample and a colonic biopsy of normal-appearing mucosa. For IBD patients, an additional biopsy was collected from visually diseased mucosa. Serum vitamin D levels were measured by ultra-performance liquid chromatography and mass spectrometry. Hematoxylin and eosin stained tissue sections from colonic biopsies were classified histologically as normal or colitis (active/inactive), and scored for the degree of inflammation present (0-3, inactive/absent to severe). Tissue sections from colonic biopsies were also stained by immunohistochemistry for VDR, for which representative diagnostic areas were photographed and scored for staining intensity using a 4-point scale. RESULTS The IBD cohort was significantly younger (40.40 ± 5.27, P < 0.05) than the non-IBD cohort (56.70 ± 1.64) with a higher prevalence of vitamin D deficiency (40% vs 20%, respectively) and insufficiency (70% vs 50%, respectively). Histologic inflammation was significantly higher in visually diseased mucosa from IBD patients (1.95 ± 0.25) than in normal-appearing mucosa from control patients (0.25 ± 0.08, P < 0.01) and from IBD patients (0.65 ± 0.36, P < 0.05) and correlated inversely with VDR expression in visually diseased colonic tissue from IBD patients (r = -0.44, P < 0.05) and from IBD patients with Crohns disease (r = -0.69, P < 0.05), but not in normal-appearing colonic tissue from control patients or IBD patients. Control and IBD patient serum vitamin D levels correlated positively with VDR expression in normal colon from control and IBD patients (r = 0.38, P < 0.05) and with patient age (r = 0.54, P < 0.01). CONCLUSION Levels of serum vitamin D correlate positively with colonic VDR expression in visually normal mucosa whereas inflammation correlates negatively with colonic VDR expression in visually diseased mucosa in Puerto Rican patients.

Immunohistochemical expression of SP-NK-1R-EGFR pathway and VDR in colonic inflammation and neoplasia

AIM To determine the expression of neurokinin-1 receptor (NK-1R), phosphorylated epidermal growth factor receptor (pEGFR), cyclooxygenase-2 (Cox-2), and vitamin D receptor (VDR) in normal, inflammatory bowel disease (IBD), and colorectal neoplasia tissues from Puerto Ricans. METHODS Tissues from patients with IBD, colitis-associated colorectal cancer (CAC), sporadic dysplasia, and sporadic colorectal cancer (CRC), as well as normal controls, were identified at several centers in Puerto Rico. Archival formalin-fixed, paraffin-embedded tissues were de-identified and processed by immunohistochemistry for NK-1R, pEGFR, Cox-2, and VDR. Pictures of representative areas of each tissues diagnosis were taken and scored by three observers using a 4-point scale that assessed intensity of staining. Tissues with CAC were further analyzed by photographing representative areas of IBD and the different grades of dysplasia, in addition to the areas of cancer, within each tissue. Differences in the average age between the five patient groups were assessed with one-way analysis of variance and Tukey-Kramer multiple comparisons test. The mean scores for normal tissues and tissues with IBD, dysplasia, CRC, and CAC were calculated and statistically compared using one-way analysis of variance and Dunnetts multiple comparisons test. Correlations between protein expression patterns were analyzed with the Pearsons product-moment correlation coefficient. Data are presented as mean ± SE. RESULTS On average, patients with IBD were younger (34.60 ± 5.81) than normal (63.20 ± 6.13, P < 0.01), sporadic dysplasia (68.80 ± 4.42, P < 0.01), sporadic cancer (74.80 ± 4.91, P < 0.001), and CAC (57.50 ± 5.11, P < 0.05) patients. NK-1R in cancer tissue (sporadic CRC, 1.73 ± 0.34; CAC, 1.57 ± 0.53) and sporadic dysplasia (2.00 ± 0.45) were higher than in normal tissues (0.73 ± 0.19). pEGFR was significantly increased in sporadic CRC (1.53 ± 0.43) and CAC (2.25 ± 0.47) when compared to normal tissue (0.07 ± 0.25, P < 0.05, P < 0.001, respectively). Cox-2 was significantly increased in sporadic colorectal cancer (2.20 ± 0.23 vs 0.80 ± 0.37 for normal tissues, P < 0.05). In comparison to normal (2.80 ± 0.13) and CAC (2.50 ± 0.33) tissues, VDR was significantly decreased in sporadic dysplasia (0.00 ± 0.00, P < 0.001 vs normal, P < 0.001 vs CAC) and sporadic CRC (0.47 ± 0.23, P < 0.001 vs normal, P < 0.001 vs CAC). VDR levels negatively correlated with NK-1R (r = -0.48) and pEGFR (r = -0.56) in normal, IBD, sporadic dysplasia and sporadic CRC tissue, but not in CAC. CONCLUSION Immunohistochemical NK-1R and pEGFR positivity with VDR negativity can be used to identify areas of sporadic colorectal neoplasia. VDR immunoreactivity can distinguish CAC from sporadic cancer.

Double immunofluorescent staining of rat macrophages in formalin-fixed paraffin-embedded tissue using two monoclonal mouse antibodies.

Abstract The conventional approach of double immunostaining to visualize more than one protein in tissues or cells using antibodies from two different host species is not always feasible due to limitations with antibody availability. Previously reported methodologies for performing multiple immunostains on the same tissue or cells with antibodies originating from the same species are varied in their complexity, sensitivity, and approach to prevent unwanted interactions between antibodies. In the ever-expanding field of macrophage biology, much more is known about mouse and human macrophages than their rat counterparts. The limited availability of validated and well-characterized monoclonal antibodies from different species is one factor responsible for preventing advances in rat macrophage biology. Here we describe an immunostaining method for identifying and examining rat macrophages that is sufficiently sensitive for use in formalin-fixed paraffin-embedded tissue and that uses only commercially available reagents and antibodies. This method can be used to help characterize both physiological and pathophysiological processes in rat macrophages and can be adapted for use with any two antibodies from the same species of origin as long as one of the antibodies is biotinylated.

Adoptive Transfer of Dendritic Cells Expressing Fas Ligand Modulates Intestinal Inflammation in a Model of Inflammatory Bowel Disease

Background Inflammatory bowel diseases (IBD) are chronic relapsing inflammatory conditions of unknown cause and likely result from the loss of immunological tolerance, which leads to over-activation of the gut immune system. Gut macrophages and dendritic cells (DCs) are essential for maintaining tolerance, but can also contribute to the inflammatory response in conditions such as IBD. Current therapies for IBD are limited by high costs and unwanted toxicities and side effects. The possibility of reducing intestinal inflammation with DCs genetically engineered to over-express the apoptosis-inducing FasL (FasL-DCs) has not yet been explored. Objective Investigate the immunomodulatory effect of administering FasL-DCs in the rat trinitrobenzene sulfonic acid (TNBS) model of acute colitis. Methods Expression of FasL on DCs isolated from the mesenteric lymph nodes (MLNs) of normal and TNBS-colitis rats was determined by flow cytometry. Primary rat bone marrow DCs were transfected with rat FasL plasmid (FasL-DCs) or empty vector (EV-DCs). The effect of these DCs on T cell IFNγ secretion and apoptosis was determined by ELISPOT and flow cytometry for Annexin V, respectively. Rats received FasL-DCs or EV-DCs intraperitoneally 96 and 48 hours prior to colitis induction with TNBS. Colonic T cell and neutrophil infiltration was determined by immunohistochemistry for CD3 and myeloperoxidase activity assay, respectively. Macrophage number and phenotype was measured by double immunofluorescence for CD68 and inducible Nitric Oxide Synthase. Results MLN dendritic cells from normal rats expressed more FasL than those from colitic rats. Compared to EV-DCs, FasL-DCs reduced T cell IFNγ secretion and increased T cell apoptosis in vitro. Adoptive transfer of FasL-DCs decreased macroscopic and microscopic damage scores and reduced colonic T cells, neutrophils, and proinflammatory macrophages when compared to EV-DC adoptive transfer. Conclusion FasL-DCs are effective at treating colonic inflammation in this model of IBD and represent a possible new treatment for patients with IBD.

The Probiotic VSL#3 Modulates Colonic Macrophages, Inflammation, and Microflora in Acute Trinitrobenzene Sulfonic Acid Colitis.

The probiotic mixture VSL#3 attenuates colitis in patients with Inflammatory Bowel Disease (IBD) and in animal models of this condition, but the mechanisms involved are incompletely understood. VSL#3 alters macrophage morphology and secretory profile in vitro in a polarization-dependent manner. We examined the effect of VSL#3 on macrophages in acute trinitrobenzene sulfonic acid-induced colitis. Rats were randomized to normal, colitis, or colitis+VSL#3 groups. After sacrifice, the colons were evaluated for macroscopic and microscopic damage. Serum cytokine levels were measured, and microbiome analysis undertaken. Total and M1 colonic macrophages, and total and proliferating hepatic macrophages were assessed by double immunofluorescence staining. Colitis+VSL#3 rats had lower macroscopic damage, with less microscopic damage in the proximal colon, compared with colitis alone. Colitis significantly increased colonic macrophage infiltration, which was significantly reduced by VSL#3 treatment. VSL#3 did not decrease the colitis-induced surge of colonic M1 macrophages or hepatic macrophages. VSL#3 reduced colitis-induced serum cytokine levels, and induced restoration of colonic transcript levels for pro-inflammatory, anti-inflammatory, and barrier proteins to, or past, normal levels. Fecal bacteria distribution changed between groups. In summary, the probiotic VSL#3 reduces colitis severity, colonic macrophage infiltration, and serum cytokine levels, but does not dampen the pro-inflammatory phenotype of M1 macrophages.