Till Örebro universitet

In this case report, we examined the levels of cytokines expressed before and during fecal stream diversion and after intestinal continuity was restored in a patient with collagenous colitis. We report the case of a 46-year-old woman with chronic, active collagenous colitis who either failed to achieve clinical remission or experienced adverse effects with the following drugs: loperamide, cholestyramine, budesonide, methotrexate and adalimumab. Due to the intractable nature of the disease and because the patient was having up to 15 watery bowel movements per day, she underwent a temporary ileostomy. Colonic biopsies were analyzed for mucosal cytokine protein levels before and during fecal stream diversion and after intestinal continuity was restored. Mucosal protein levels of interleukin (IL)-1β, IL-2, IL-6, IL-12, IL-17 A, IL-23, TNF, IFN-γ, IL-4, IL-5, IL-10 and IL-13 were all higher during active disease and decreased to non-detectable or considerably lower levels during fecal stream diversion. One month after the restoration of bowel continuity, when the patient experienced a relapse of symptoms, IL-2, IL-23 and IL-21 levels were again increased. Our results indicate that fecal stream diversion in this patient suppressed the levels of all cytokines analyzed in colonic biopsies. With the recurrence of clinical symptoms and histological changes after bowel reconstruction, the levels of primarily proinflammatory cytokines increased. Our findings support the hypothesis that a luminal factor triggers the inflammation observed in collagenous colitis.

Background: All-trans retinoic acid (atRA) plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development but may in turn be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis. The cellular atRA levels are under strict control involving several cytochromes P450 isoforms (CYPs). CYP26 may be the most important regulator of atRA catabolism in vascular cells. The present study describes the molecular cloning, characterization and function of atRA-induced expression of a spliced variant of the CYP26B1 gene.

Methodology/Principal Findings: The coding region of the spliced CYP26B1 lacking exon 2 was amplified from cDNA synthesized from atRA-treated human aortic smooth muscle cells and sequenced. Both the spliced variant and full length CYP26B1 was found to be expressed in cultured human endothelial and smooth muscle cells, and in normal and atherosclerotic vessel. atRA induced both variants of CYP26B1 in cultured vascular cells. Furthermore, the levels of spliced mRNA transcript were 4.5 times higher in the atherosclerotic lesion compared to normal arteries and the expression in the lesions was increased 20-fold upon atRA treatment. The spliced CYP26B1 still has the capability to degrade atRA, but at an initial rate one-third that of the corresponding full length enzyme. Transfection of COS-1 and THP-1 cells with the CYP26B1 spliced variant indicated either an increase or a decrease in the catabolism of atRA, probably depending on the expression of other atRA catabolizing enzymes in the cells.

Conclusions/Significance: Vascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions. The spliced variant displays a slower and reduced degradation of atRA as compared to the fulllength enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1 splice variant in health and disease.

Background

All-trans retinoic acid (atRA) plays an essential role in the regulation of gene expression, cell growth and differentiation and is also important for normal cardiovascular development but may in turn be involved in cardiovascular diseases, i.e. atherosclerosis and restenosis. The cellular atRA levels are under strict control involving several cytochromes P450 isoforms (CYPs). CYP26 may be the most important regulator of atRA catabolism in vascular cells. The present study describes the molecular cloning, characterization and function of atRA-induced expression of a spliced variant of the CYP26B1 gene.

Methodology/Principal Findings

The coding region of the spliced CYP26B1 lacking exon 2 was amplified from cDNA synthesized from atRA-treated human aortic smooth muscle cells and sequenced. Both the spliced variant and full length CYP26B1 was found to be expressed in cultured human endothelial and smooth muscle cells, and in normal and atherosclerotic vessel. atRA induced both variants of CYP26B1 in cultured vascular cells. Furthermore, the levels of spliced mRNA transcript were 4.5 times higher in the atherosclerotic lesion compared to normal arteries and the expression in the lesions was increased 20-fold upon atRA treatment. The spliced CYP26B1 still has the capability to degrade atRA, but at an initial rate one-third that of the corresponding full length enzyme. Transfection of COS-1 and THP-1 cells with the CYP26B1 spliced variant indicated either an increase or a decrease in the catabolism of atRA, probably depending on the expression of other atRA catabolizing enzymes in the cells.

Conclusions/Significance

Vascular cells express the spliced variant of CYP26B1 lacking exon 2 and it is also increased in atherosclerotic lesions. The spliced variant displays a slower and reduced degradation of atRA as compared to the full-length enzyme. Further studies are needed, however, to clarify the substrate specificity and role of the CYP26B1 splice variant in health and disease.

Microscopic colitis (MC), comprising collagenous colitis (CC) and lymphocytic colitis (LC), is a common cause of chronic diarrhea. Various immune cell infiltrations in the epithelium and lamina propria are seen in MC immunopathology. We compared gene and protein expressions of different immune cell attracting chemokines and their receptors in colon biopsies from MC patients in active disease or histopathological remission (CC/LC-HR) with controls, using qRT-PCR and Luminex, respectively. CC and LC patients with active disease demonstrated a mixed chemokine profile with significantly enhanced gene and/or protein expressions of the chemokines CCL2, CCL3, CCL4, CCL5, CCL7, CCL22, CXCL8, CXCL9, CXCL10, CXCL11, and CX(3)CL1 and the receptors CCR2, CCR3, CCR4, CXCR1, CXCR2, and CX(3)CR1. Enhanced chemokine/chemokine receptor gene and protein levels in LC-HR patients were similar to LC patients, whereas CC-HR patients demonstrated almost normalized levels. These findings expand the current understanding of the involvement of various immune cells in MC immunopathology and endorse chemokines as potential diagnostic markers as well as therapeutic candidates. Moreover, this study further supports the hypothesis that CC and LC are two different entities due to differences in their immunoregulatory responses.

Background and Aims: Microscopic colitis (MC), encompassing the subgroups collagenous colitis (CC) and lymphocytic colitis (LC), is characterized by macroscopically normal or near-normal colonic mucosa, and an increased number of intraepithelial lymphocytes (IELs) and mononuclear cell infiltration in the underlying lamina propria (LP), in addition to an increased collagen layer in CC. This study aimed to characterize the inflammatory cells involved in mucosal inflammation, using immunohistochemistry.

Methods Paraffin-embedded biopsies from 23 untreated patients with MC (CC = 13, LC = 10) and 17 controls were stained with antibodies against CD3, CD4, CD8, CD20, CD30, Foxp3, CD45RO and Ki67. Computerized image analysis was used to calculate areas of stained lymphocytes in the surface and crypt epithelia as well as in the LP.

Results In CC and LC, an increase of predominantly CD8+ lymphocytes was seen in both the epithelium and the lamina propria, whereas a decreased amount of CD4+ lymphocytes was found in the lamina propria. CD45RO+ and Foxp3+ cells were more abundant in all areas in both patient groups compared to controls, as were CD20+ areas, although more scarce. Ki67+ areas were only more abundant in the epithelium, whereas CD30+ areas were more abundant in the lamina propria of both patient groups compared to controls.

Conclusions This study confirms an increased amount of CD8+ lymphocytes in the epithelium. Lymphocytic proliferation and activation markers were more abundant, whereas a decreased amount of CD4+ lymphocytes was seen in the LP. Further studies are needed to reveal the underlying mechanism(s).

Gαi2-deficient mice spontaneously develop colitis. Using xMAP technology and RT-PCR, we investigated cytokine/chemokine profiles during histologically defined phases of disease: (i) no/mild, (ii) moderate, (iii) severe colitis without dysplasia/cancer and (iv) severe colitis with dysplasia/cancer, compared with age-matched wild-type (WT) littermates. Colonic dysplasia was observed in 4/11 mice and cancer in 1/11 mice with severe colitis. The histology correlated with progressive increases in colon weight/cm and spleen weight, and decreased thymus weight, all more advanced in mice with dysplasia/cancer. IL-1β, IL-6, IL-12p40, IL-17, TNF-α, CCL2 and CXCL1 protein levels in colons, but not small intestines increased with colitis progression and were significantly increased in mice with moderate and severe colitis compared with WT mice, irrespective of the absence/presence of dysplasia/cancer. CCL5 did not change during colitis progression. Colonic IL-17 transcription increased 40- to 70-fold in all stages of colitis, whereas IFN-γ mRNA was gradually up-regulated 12- to 55-fold with colitis progression, and further to 62-fold in mice with dysplasia/cancer. IL-27 mRNA increased 4- to 15-fold during the course of colitis, and colonic IL-21 transcription increased 3-fold in mice with severe colitis, both irrespective of the absence/presence of dysplasia/cancer. FoxP3 transcription was significantly enhanced (3.5-fold) in mice with moderate and severe colitis, but not in mice with dysplasia/cancer, compared with WT mice. Constrained correspondence analysis demonstrated an association between increased protein levels of TNF-α, CCL2, IL-1β, IL-6 and CXCL1 and dysplasia/cancer. In conclusion, colonic responses are dominated by a mixed T(h)1/T(h)17 phenotype, with increasing T(h)1 cytokine transcription with progression of colitis in Gαi2(-/-) mice.

AIM: To investigate Toll-like receptor (TLR) signaling regulators in microscopic and ulcerative colitis patients.

METHODS: Total RNA and microRNA were isolated from fresh frozen colonic biopsies of non-inflamed controls and patients with active or in-remission collagenous colitis (CC), lymphocytic colitis (LC), or ulcerative colitis (UC). We compared expressions of interleukin-1 receptor-associated kinase (IRAK)-2, IRAK-M, interleukin (IL)-37, microRNA (miR)-146a, miR-155, and miR-21 using quantitative real time reverse transcription polymerase chain reaction.

RESULTS: IRAK-M expression was increased in LC patients with active disease in histopathological remission (LC-HR; P = 0.02) and UC patients (P = 0.01), but no differences in IRAK-2 expression were detected compared to controls. miR-146a, -155 and -21 expressions were increased in LC-HR (P = 0.04, 0.07, and 0.004) and UC (P = 0.02, 0.04 and 0.03) patients. miR-146a and miR-21 expressions were significantly enhanced in UC patients compared to UC remission (UC-R; P = 0.01 and 0.04). Likewise, active CC patients showed significantly increased expression of miR-155 (P = 0.003) and miR-21 (P = 0.006). IL-37 expression was decreased in both CC (P = 0.03) and LC (P = 0.04) patients with a similar trend in UC patients but not statistically significant, whilst it was increased in UC-R patients compared to controls (P = 0.02) and active UC (P = 0.001).

CONCLUSION: The identification of differentially expressed miRNAs, IL-37, and IRAK-M suggests different pathophysiologic mechanisms in various disease stages in LC, CC, and UC. (C) 2014 Baishideng Publishing Group Inc. All rights reserved.

In atherosclerotic lesions, monocyte-derived macrophages are major source of interferon gamma (IFN-γ), a pleotropic cytokine known to regulate the expression of numerous genes, including the antiviral gene RSAD2. While RSAD2 was reported to be expressed in endothelial cells of human carotid lesions, its significance for the development of atherosclerosis remains utterly unknown. Here, we harnessed publicly available human carotid atherosclerotic data to explore RSAD2 in lesions and employed siRNA-mediated gene-knockdown to investigate its function in IFN-γ-stimulated human aortic smooth muscle cells (hAoSMCs). Silencing RSAD2 in IFN-γ-stimulated hAoSMCs resulted in reduced expression and secretion of key CXCR3-chemokines, CXCL9, CXCL10, and CXCL11. Conditioned medium from RSAD2-deficient hAoSMCs exhibited diminished monocyte attraction in vitro compared to conditioned medium from control cells. Furthermore, RSAD2 transcript was elevated in carotid lesions where it was expressed by several different cell types, including endothelial cells, macrophages and smooth muscle cells. Interestingly, RSAD2 displayed significant correlations with CXCL10 (r =  0.45, p = 0.010) and CXCL11 (r = 0.53, p = 0.002) in human carotid lesions. Combining our findings, we uncover a novel role for RSAD2 in hAoSMCs, which could potentially contribute to monocyte recruitment in the context of atherosclerosis.

Background and Aims: During atherosclerosis, activated vascular cells secrete chemokines to recruit leukocytes to the subendothelial space. Activated leukocytes, in particular activated T lymphocytes express IFN-γ, which affects the expression of numerous genes, including the antiviral protein viperin, in vascular cells. Viperin is expressed in endothelial cells of human carotid plaques but its relevance for vascular physiology and/or pathophysiology has not been established (Olofsson et al., 2005). We aimed at studying the function of viperin in human AoSMCs.

Methods: Immunostaining of human carotid plaques was performed to determine the expression and localization of viperin in carotid plaques. To study the role of viperin in regulation of vascular inflammation, viperin was silenced using siRNA in cultured human AoSMCs, prior to stimulation with IFN-γ. Release of inflammatory mediators was analyzed using OLINK proteomics and ELISA, and the gene expression was analyzed by qRT-PCR.

Results: Viperin is expressed in endothelial cells, macrophages and smooth muscle cells in human carotid plaques. OLINK data analysis revealed reduction in release of CCL3, CXCL9, CXCL10 and CXCL11 by AoSMCs that were targeted with anti-viperin siRNA prior to stimulation with IFN-γ. Using ELISA and qRT-PCR, we could confirm the reduction of IFN-γ-induced CXCL10 and CXCL11 in viperin knockdown AoSMCs.

Conclusions: Viperin is expressed in human carotid plaques and seem to be involved in regulating the expression of CXCL10 and CXCL11 in AoSMCs. Thus, it may play a role in recruitment of T lymphocytes to the subendothelial space in atherosclerotic plaques.

Laminins are essential components of the basement membranes, expressed in a tissue- and cell-specific manner under physiological conditions. During inflammatory circumstances, such as atherosclerosis, alterations in laminin composition within vessels have been observed. Our study aimed to assess the influence of tumor necrosis factor-alpha (TNF), a proinflammatory cytokine abundantly found in atherosclerotic lesions, on endothelial laminin gene expression and the effects of laminin-332 (LN332) on endothelial cells' behavior. We also evaluated the expression of LN332-encoding genes in human carotid atherosclerotic plaques. Our findings demonstrate that TNF induces upregulation of LAMB3 and LAMC2, which, along with LAMA3, encode the LN332 isoform. Endothelial cells cultured on recombinant LN332 exhibit decreased claudin-5 expression and display a loosely connected phenotype, with an elevated expression of chemokines and leukocyte adhesion molecules, enhancing their attractiveness and adhesion to leukocytes in vitro. Furthermore, LAMB3 and LAMC2 are upregulated in human carotid plaques and show a positive correlation with TNF expression. In summary, TNF stimulates the expression of LN332-encoding genes in human endothelial cells and LN332 promotes an endothelial phenotype characterized by compromised junctional integrity and increased leukocyte interaction. These findings highlight the importance of basement membrane proteins for endothelial integrity and the potential role of LN332 in atherosclerosis.

Objectives: Faecal microbiota transfer (FMT) consists of the infusion of donor faecal material into the intestine of patients with the aim to restore a disturbed gut microbiota.

Methods: In this pilot study (NCT03275467), the effect of three repeated FMTs (day 0, two weeks, four weeks) was studied and followed up for six months in nine collagenous colitis (CC) patients, using two stool donors.

Results: Five patients had an active disease at the time of baseline sampling. The primary endpoint (remission at six weeks, defined as <3 stools whereof <1 watery stool per day) was achieved by two of these patients, and by one at eight weeks. Overall, in all nine patients, FMT did not result in a significant reduction of watery stools, assessed by daily diary. However, diarrhoea (assessed by gastrointestinal symptom rating scale) was significantly improved at four (p = .038) and eight weeks (p = .038), indigestion at eight (p = .045) and 12 weeks (p = .006), disease-related worries at four (p = .027) and eight weeks (p = .027), and quality of life at six months (p = .009). FMT resulted in an increased number of lamina propria lymphocytes, possibly indicating an initial mucosal immune activation. No serious adverse events, no systemic effects, and no changes in faecal calprotectin and psychological symptoms were observed.

Conclusions: FMT is able to improve symptoms in a yet undefined subset of CC patients. Further studies could help to characterise this subset and to understand if these results can be generalised to all microscopic colitis patients.

Trimethylamine-N-oxide (TMAO) is a gut microbiota-derived metabolite and TNF-α is proinflammatory cytokine, both known to be associated with renal inflammation, fibrosis and chronic kidney disease. However, today there are no data showing the combined effect of TMAO and TNF-α on renal fibrosis-and inflammation. The aim of this study was to investigate whether TMAO can enhance the inflammatory and fibrotic effects of TNF-α on renal fibroblasts. We found that the combination of TNF-α and TMAO synergistically increased fibronectin release and total collagen production from renal fibroblasts. The combination of TMAO and TNF-α also promoted increased cell proliferation. Both renal proliferation and collagen production were mediated through Akt/mTOR/ERK signaling. We also found that TMAO enhanced TNF-α mediated renal inflammation by inducing the release of several cytokines (IL-6, LAP TGF-beta-1), chemokines (CXCL-6, MCP-3), inflammatory-and growth mediators (VEGFA, CD40, HGF) from renal fibroblasts. In conclusion, we showed that TMAO can enhance TNF-α mediated renal fibrosis and release of inflammatory mediators from renal fibroblasts in vitro. Our results can promote further research evaluating the combined effect of TMAO and inflammatory mediators on the development of kidney disease.

Trimethylamine N-oxide (TMAO), a product of gut microbiota metabolism, has previously been shown to be implicated in chronic kidney disease. A high TMAO-containing diet has been found to cause tubulointerstitial renal fibrosis in mice. However, today there are no data linking specific molecular pathways with the effect of TMAO on human renal fibrosis. The aim of this study was to investigate the fibrotic effects of TMAO on renal fibroblasts and to elucidate the molecular pathways involved. We found that TMAO promoted renal fibroblast activation and fibroblast proliferation via the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 signaling. We also found that TMAO increased the total collagen production from renal fibroblasts via the PERK/Akt/mTOR pathway. However, TMAO did not induce fibronectin or TGF-β1 release from renal fibroblasts. We have unraveled that the PERK/Akt/mTOR pathway, NLRP3, and caspase-1 mediates TMAO's fibrotic effect on human renal fibroblasts. Our results can pave the way for future research to further clarify the molecular mechanism behind TMAO's effects and to identify novel therapeutic targets in the context of chronic kidney disease.

Trimethylamine-N-oxide (TMAO) is a uremic toxin, which has been associated with chronic kidney disease (CKD). Renal tubular epithelial cells play a central role in the pathophysiology of CKD. Megalin is an albumin-binding surface receptor on tubular epithelial cells, which is indispensable for urine protein reabsorption. To date, no studies have investigated the effect of TMAO on megalin expression and the functional properties of human tubular epithelial cells. The aim of this study was first to identify the functional effect of TMAO on human renal proximal tubular cells and second, to unravel the effects of TMAO on megalin-cubilin receptor expression. We found through global gene expression analysis that TMAO was associated with kidney disease. The microarray analysis also showed that megalin expression was suppressed by TMAO, which was also validated at the gene and protein level. High glucose and TMAO was shown to downregulate megalin expression and albumin uptake similarly. We also found that TMAO suppressed megalin expression via PI3K and ERK signaling. Furthermore, we showed that candesartan, dapagliflozin and enalaprilat counteracted the suppressive effect of TMAO on megalin expression. Our results may further help us unravel the role of TMAO in CKD development and to identify new therapeutic targets to counteract TMAOs effects.

Microscopic colitis (MC) is a chronic diarrhoeal disease of unknown aetiology, comprising collagenous colitis (CC) and lymphocytic colitis (LC). The nature of the adaptive local immune responses in the mucosa of MC patients is however far from elucidated. The present study investigates phenotypic and functional characteristics of the adaptive local immune responses in the colonic mucosa of these patients.

Our immunohistochemistry and flow cytometry studies (Paper I & II) demonstrated increased frequencies of CD8+ T cells in the colonic epithelium and lamina propria of both LC and CC patients compared to controls, whereas the frequencies of CD4+ T cells were unaltered or reduced. Our flow cytometry data revealed increased local activation of both CD4+ and CD8+ T cells in the lamina propria as well as the intraepithelial compartment of CC and LC patients compared to controls, demonstrated as increased proportions of these cells expressing the active/memory marker CD45RO and the proliferation marker Ki67.

Analysis of recent thymic emigrants by measuring T cell receptor excision circle (TREC) levels in the colonic mucosa of CC and LC patients revealed reduced TRECs levels in these patients compared to controls (Paper III). These results suggests that the observed increased numbers of T cells in the mucosa of CC and LC patients is due to the expansion of local resident T cells rather than direct recruitment of recent thymic emigrants to the mucosa.

Molecular analysis of T helper (Th) cell and cytotoxic T lymphocyte (Tc) mucosal cytokines at messenger and protein levels in the colonic biopsies from CC and LC patients demonstrated a mixed Th17/Tc17 and Th1/Tc1 mucosal cytokine profile and revealed significant differences in the mucosal cytokine levels in CC and LC patients compared to controls (Paper IV).

Finally, we have set up an in vitro model to investigate how the colonic milieu affects the activation and differentiation of T lymphocytes (Paper V). Our preliminary data indicate increased production of both pro inflammatory and antiinflammatory cytokines by peripheral blood T cells in the presence of soluble factors from the inflamed colonic mucosa of CC patients compared to controls.

Dysregulated T cell responses in the intestine may lead to chronic bowel inflammation such as collagenous colitis (CC) and lymphocytic colitis (LC), together known as microscopic colitis (MC). Having demonstrated increased local T cell responses in the intestinal mucosa of MC patients, we investigated the recent thymic emigrants by measuring T cell receptor excision circle (TREC) levels in the colonic biopsies from CC (n = 8), LC (n = 5), and CC or LC patients in histopathological remission (CC-HR, n = 3) (LC-HR, n = 6), non-inflamed diarrhoea patients (n = 17), and controls (n = 10) by real-time PCR. We observed lower median TREC levels in both CC and LC patients as well as in LC-HR patients compared to controls. In contrast to MC patients, non-inflamed diarrhoea patients presented with enhanced TREC levels compared to controls. None of the recorded differences did, however, reach statistical significance. A trend towards increased relative expression of CD3 was noted in all MC subgroups examined and reached statistical significance in LC patients compared to controls. In conclusion, reduced TRECs level in the colonic mucosa, together with our previously demonstrated enhanced expression of Ki67(+) T cells, suggests local expansion of resident T lymphocytes in the inflamed mucosa of MC patients.

Aims: Dysregulated T cell responses in the intestine may lead to chronic bowel inflammation such as collagenous colitis (CC) and lymphocytic colitis (LC), together known as microscopic colitis (MC). Having demonstrated increased local T cell responses in the intestinal mucosa of MC patients, we investigated the recent thymic emigrants by measuring T cell receptor excision circle (TREC) levels in the colonic mucosa of CC and LC patients.

Methods: Mucosal biopsies from CC (n=8), LC (n=5), and CC or LC patients in histopathological remission, (CC-HR, n=3), (LC-HR, n=6), non-inflamed diarrhoea patients (n=17) and controls (n=10) were analysed for TRECs expression by real time PCR.

Results: The median TREC levels were lower in both CC and LC patients as well as in LCHR patients compared to controls. In contrast to MC patients, non-inflamed diarrhoea patients presented with enhanced TREC levels compared to controls. None of the recorded differences did however reach statistical significance. No differences were observed in median TREC levels in either CC-HR or LC-HR patients compared to active CC and LC patients. A trend towards increased relative expression of CD3 was noted in all MC subgroups examined; and reached statistical significance in LC patients compared to controls. LC patients had ignificantly increased CD3 mRNA levels also compared to CC, CC-HR, LC-HR and non-inflamed iarrhoea patients.

Conclusions: Reduced TRECs level in the colonic mucosa, together with our previously demonstrated enhanced expression of Ki67⁺ T cells, suggest local expansion of resident T lymphocytes in the inflamed mucosa of MC patients.

Soluble factors from intestinal mucosal cells contribute to immune homeostasis in the gut. We have established an in vitro model to investigate the regulatory role of soluble factors from inflamed intestinal mucosa of collagenous colitis (CC) patients in the differentiation of T cells. Peripheral blood CD4(+) T cells from healthy donors were polyclonally activated in the presence of conditioned medium (CM) generated from denuded biopsies (DNB) or isolated lamina propria mononuclear cells (LPMCs) from mucosal biopsies from CC patients compared to noninflamed controls, to determine proliferation and secretion of cytokines involved in T-cell differentiation. Compared to controls, we observed significantly increased production of the proinflammatory cytokines IFN-gamma, IL-17A, IL-6, and IL-1 beta and the anti-inflammatory cytokines IL-4 and IL-10 in the presence of CC-DNB-CM. The most pronounced effect of CC-LPMC-CM on peripheral CD4(+) T cells was a trend towards increased production of IL-17A and IL-10. A trend towards reduced inhibition of T-cell proliferation was noted in the presence of CC-DNB-CM. In conclusion, our in vitro model reveals implications of soluble factors from CC colonic mucosa on peripheral T cells, enhancing their production of both pro-and anti-inflammatory cytokines.

Understanding how uropathogenic Escherichia coli (UPEC) modulates the immune response in the kidney is essential to prevent UPEC from reaching the bloodstream and causing urosepsis. The purpose of this study was to elucidate if renal fibroblasts can release IL-1β during a UPEC infection and to investigate the mechanism behind the IL-1β release. We found that the UPEC strain CFT073 induced an increased IL-1β and LDH release from renal fibroblasts, but not from renal epithelial cells. The UPEC-induced IL-1β release was found to be NLRP3, caspase-1, caspase-4, ERK 1/2, cathepsin B and serine protease dependent in renal fibroblasts. We also found that the UPEC virulence factor α-hemolysin was necessary for IL-1β release. Conditioned medium from caspase-1, caspase-4 and NLRP3-deficient renal fibroblasts mediated an increased reactive oxygen species production from neutrophils, but reduced UPEC phagocytosis. Taken together, our study demonstrates that renal fibroblasts, but not renal epithelial cells, release IL-1β during a UPEC infection. This suggest that renal fibroblasts are vital immunoreactive cells and not only structural cells that produce and regulate the extracellular matrix.

Background: Collagenous colitis (CC) and lymphocytic colitis (LC) are chronic inflammatory bowel disorders of unknown etiology. This study investigated phenotypic characteristics of the mucosal lymphocytes in CC and LC.

Methods: Lamina propria and intraepithelial lymphocytes (LPLs, IELs) isolated from mucosal biopsies from CC (n = 7), LC (n = 6), as well as LC or CC patients in histopathological remission, (LC-HR) (n = 6) and CC-HR (n = 4) and non-inflamed controls (n = 10) were phenotypically characterized by four-color flow cytometry.

Results: The proportions of CD8+ IELs were increased in CC and LC (p < 0.01) compared to controls. Increased proportions of CD45RO+CD8+ IELs and LPLs were observed in LC and even more in CC patients (p < 0.01). Both CC (p < 0.05) and LC patients had elevated proportions of CD4+8+ IELs and LPLs compared to controls. The proportions of CD45RO+ cells were increased in CD4+8+ IELs and LPLs (p < 0.05) in CC and LC patients compared to controls. Both CC (p < 0.05) and LC patients had higher proportions of Ki67+CD8+ IELs and LPLs compared to controls.

In contrast, decreased proportions of CD4+ LPLs were observed in CC and LC as well as CD4+ IELs in LC compared to controls. Increased proportions of Ki67+CD4+ IELs and LPLs (p < 0.05) were observed in CC and LC patients. CC-HR but not LC-HR patients demonstrated normalized proportions of both IELs and LPLs compared to CC and LC patients respectively.

Conclusion: LC and CC patients have differences in mucosal lymphocyte subsets, with increased proportions of Ki67+ and CD45RO+ CD8+ and CD4+8+ mucosal T cells.

Background:

Microscopic colitis (MC) is a chronic inflammatory bowel disorder of unknown aetiology comprising collagenous colitis (CC) and lymphocytic colitis (LC). Data on the local cytokine profile in MC is limited. This study investigated the T helper (Th) cell and cytotoxic T lymphocyte (CTL) mucosal cytokine profile at messenger and protein levels in MC patients.

Methods:

Mucosal biopsies from CC (n = 10), LC (n = 5), and CC or LC patients in histopathological remission (CC-HR, n = 4), (LC-HR, n = 6), ulcerative colitis (UC, n = 3) and controls (n = 10) were analysed by real-time PCR and Luminex for expression/production of IL-1 beta, -4, -5, -6, -10, -12, -17, -21, -22, -23, IFN-gamma, TNF-alpha, T-bet and RORC2.

Results:

Mucosal mRNA but not protein levels of IFN-gamma and IL-12 were significantly up regulated in CC, LC as well as UC patients compared to controls. Transcription of the Th1 transcription factor T-bet was significantly enhanced in CC but not LC patients. mRNA levels for IL-17A, IL-21, IL-22 and IL-6 were significantly up regulated in CC and LC patients compared to controls, albeit less than in UC patients. Significantly enhanced IL-21 protein levels were noted in both CC and LC patients. IL-6 protein and IL-1 beta mRNA levels were increased in CC and UC but not LC patients. Increased mucosal mRNA levels of IFN-gamma, IL-21 and IL-22 were correlated with higher clinical activity, recorded as the number of bowel movements per day, in MC patients.

Although at lower magnitude, IL-23A mRNA was upregulated in CC and LC, whereas TNF-alpha protein was increased in CC, LC as well as in UC patients.

Neither mRNA nor protein levels of IL-4, IL-5 or IL-10 were significantly changed in any of the colitis groups. LC-HR and especially CC-HR patients had normalized mRNA and protein levels of the above cytokines compared to LC and CC patients. No significant differences were found between LC and CC in cytokine expression/production.

Conclusion:

LC and CC patients demonstrate a mixed Th17/Tc17 and Th1/Tc1 mucosal cytokine profile.

Background: Soluble factors released by intestinal mucosal cells contribute to immune homeostasis in the gut. This is the first study to investigate the role of soluble factors from the intestinal mucosa of collagenous colitis (CC) patients in the regulation of effector T cells using a novel system that mimics the in vivo exposure of newly recruited peripheral blood T cells to soluble factors derived from the colonic milieu of normal individuals and inflamed CC patient mucosa.

Methods: Denuded biopsies (DNB) and isolated lamina propria mononuclear cells (LPMCs) from mucosal biopsies from CC patients and non-inflamed controls were cultured to collect conditioned medium (CM). Enriched peripheral blood CD4⁺ T cells from healthy donors were polyclonally activated in the absence or presence of CM from CC patients and controls. Proliferation, as well as secretion of IL-1β IL-4, IL-6, IL-10, IL-17A, IFN-γ and TNF-α was analysed the latter with Luminex® analysis.

Results: Peripheral CD4⁺ T cells exposed to CM from the colonic mucosa demonstrated reduced proliferation. This inhibition was less pronounced with DNB-CM derived from CC patients compared to non-inflamed control mucosa. In contrast, LPMC-CM from non-inflamed controls inhibited T-cell proliferation less than LPMC-CM from CC patients. Both DNB-CM and LPMC-CM from CC patients induced more or less increased production of the proinflammatory cytokines IFN-γ, IL-17A, IL-6 and TNF-α as well as the anti-inflammatory cytokines IL-4 and IL-10 from peripheral CD4⁺ T cells compared to non-inflamed controls. In contrast, IL-1β production by peripheral T cells showed mixed results – it was either increased or reduced in the presence of both DNB and LPMC-CM from CC patients compared to noninflamed controls with different blood donors and different concentrations.

Conclusion: Our preliminary data indicates reduced inhibition of proliferation of peripheral CD4⁺ T cells in the presence of mucosa-derived soluble factors from CC patients compared to controls. In addition, increased production of both inflammatory and anti-inflammatory cytokines by peripheral CD4⁺ T cells was recorded in the presence of soluble factors from the colonic mucosa of CC patients compared to controls. This model can be valuable in evaluating the effect(s) of existing and new drugs on T cell differentiation in the intestinal mucosa.

Macrophages play a crucial role in maintaining homeostasis in the intestine, but the underlying mechanisms have not yet been elucidated fully. Here we show for the first time that mature intestinal macrophages in mouse colon and small intestine express high levels of αvβ5 integrin, which acts as a receptor for the uptake of apoptotic cells and can activate molecules involved in several aspects of tissue homeostasis such as angiogenesis and remodelling of the extracellular matrix. αvβ5 is not expressed by other immune cells in the intestine, is already present on intestinal macrophages soon after birth, and its expression is not dependent on the microbiota. In adults, αvβ5 induces the differentiation of monocytes in response to the local environment and it confers intestinal macrophages with the ability to promote engulfment of apoptotic cells via engagement of the bridging molecule milk fat globule EGF-like molecule 8. In the absence of αvβ5, there are fewer monocytes in the mucosa and mature intestinal macrophages have decreased expression of metalloproteases and interleukin 10. Mice lacking αvβ5 on haematopoietic cells show increased susceptibility to chemical colitis and we conclude that αvβ5 contributes to the tissue repair by regulating the homeostatic properties of intestinal macrophages.

The balance between pro- and anti-inflammatory cytokines released by immune and non-immune cells plays a decisive role in the progression of atherosclerosis. Interleukin (IL)-17A has been shown to accelerate atherosclerosis. In this study, we investigated the effect on pro-inflammatory mediators and atherosclerosis development of an Affibody molecule that targets IL17A. Affibody molecule neutralizing IL17A, or sham were administered in vitro to human aortic smooth muscle cells (HAoSMCs) and murine NIH/3T3 fibroblasts and in vivo to atherosclerosis-prone, hyperlipidaemic ApoE(-/-) mice. Levels of mediators of inflammation and development of atherosclerosis were compared between treatments. Exposure of human smooth muscle cells and murine NIH/3T3 fibroblasts in vitro to alpha IL-17A Affibody molecule markedly reduced IL6 and CXCL1 release in supernatants compared with sham exposure. Treatment of ApoE(-/-) mice with alpha IL-17A Affibody molecule significantly reduced plasma protein levels of CXCL1, CCL2, CCL3, HGF, PDGFB, MAP2K6, QDPR, and splenocyte mRNA levels of Ccxl1, Il6, and Ccl20 compared with sham exposure. There was no significant difference in atherosclerosis burden between the groups. In conclusion, administration of alpha IL17A Affibody molecule reduced levels of pro-inflammatory mediators and attenuated inflammation in ApoE(-/-) mice.

Background and Aims: Peripheral arterial disease (PAD) is a clinical manifestation of atherosclerosis, affecting arteries in the leg. Based on their symptoms and severity, PAD patients are characterized into three sub-groups: asymptomatic, intermittent claudication (IC) and critical limb ischemia (CLI). Despite its high prevalence, PAD remains under diagnosed and the role of immune cells in PAD pathophysiology remains poorly understood. In this study, we characterized the innate immune responses in PAD patients compared to healthy controls.

Methods: Blood samples were collected from 14 patients with PAD (IC) and 30 healthy controls, to assess the phenotype of monocytes and neutrophils by using 10-colour flow cytometry. Phagocytosis assay was performed with labelled E.coli particles. Mann-Whitney U non-parametrical test was used for statistical comparison between PAD patients and healthy controls.

Results: A significant higher proportion of leukocytes (p<0.05) and neutrophils (p<0.01) was observed in PAD patients compared to healthy controls, whereas monocyte subsets showed no significant differences. Interestingly, neutrophils showed a significantly impaired phagocytosis capability (p<0.05) and reduced expression of myeloperoxidase (MPO) (p<0.05) in PAD patients compared to healthy controls.

Conclusions: Taken together these results, suggest that PAD patients have an increased proportion of neutrophils in circulation, with impaired phagocytosis capability, compared to healthy controls.

Background and Aims: Peripheral arterial disease (PAD) is a common manifestation of atherosclerosis, affecting the lower limbs. T cells are among the principal contributors to the development of atherosclerotic plaques. However, T cell immune responses in PAD pathophysiology are poorly understood and a detailed phenotypic and functional characterization of T cell immune responses in PAD is needed.

Methods: Blood samples were collected from PAD patients with claudicatio intermittens (n=14) and healthy controls (HCs, n=30). We assessed the phenotype of active, effector and memory T cell subsets by evaluating the expression of specific surface and intracellular markers analysed by 10-colour flow cytometry. Functional responses were evaluated by performing T cell receptor (TCR) stimulation of PBMCs in a 3D cell culture system to assess cytokine production by ELISA. Statistical analyses were performed using the Mann-Whitney U test.

Results: No differences were observed between PAD and HCs in terms of active, effector and memory T cell phenotypes and in the frequency of cells expressing CCR6 and CXCR3 (markers associated with T cells producing IL-17 and IFN-γ). However, lower frequencies of IFN-γ+ cells among CD8+ (P=0.04), and CD4+CD8+ cells (P=0.03) were observed in PAD compared to HCs. TNF-α production in PAD-derived PBMCs, via TCR stimulation was increased at both 48- (P=0.004) and 72-hour time points (P=0.003). No differences were observed in IL-1β, IFN-γ and IL-17 secretion.

Conclusions: Taken together these results suggest that increased TNF-α secretion by PBMCs in response to TCR activation might contribute to the pro-inflammatory environment in PAD pathogenesis.

Ageing is associated with dysregulated immune responses, resulting in impaired resilience against infections and low-grade inflammation known as inflammageing. Frailty is a measurable condition in older adults characterized by decreased health and physical impairment. Dendritic cells (DCs) and monocytes play a crucial role in initiating and steering immune responses. To assess whether their frequencies and phenotypes in the blood are affected by ageing or frailty, we performed a flow cytometry study on monocyte and DC subsets in an immune ageing cohort. We included (n = 15 in each group) healthy young controls (HYC, median age 29 years), healthy older controls (HOC, 73 years) and Frail older controls (76 years). Monocyte subsets (classical, intermediate, non-classical) were identified by CD14 and CD16 expression, and DC subsets (conventional (c)DC1, cDC2, plasmacytoid (p)DC) by CD11c, CD1c, CD141 and CD303 expression. All subsets were checked for TLR2, TLR4, HLA-DR, CD86, PDL1, CCR7 and CD40 expression. We observed a lower proportion of pDCs in HOC compared to HYC. Additionally, we found higher expression of activation markers on classical and intermediate monocytes and on cDC2 in HOC compared to HYC. Frail participants had a higher expression of CD40 on classical and non-classical monocytes compared to the HOC group. We document a substantial effect of ageing on monocytes and DCs. Reduced pDCs in older people may underlie their impaired ability to counter viral infections, whereas enhanced expression of activation markers could indicate a state of inflammageing. Future studies could elucidate the functional consequences of CD40 upregulation with frailty.

Background: Irritable bowel syndrome (IBS) is characterized by chronic abdominal symptoms such as pain, discomfort, and altered bowel habits. A subset of IBS patients, denoted as post-infectious IBS (PI-IBS) patients, develop symptoms after an enteric infection. Distinct abnormalities in the gut mucosa, including mucosal inflammation, have been proposed to contribute to or be the cause of PI-IBS. This study investigated lymphocyte subsets in PI-IBS patients compared to healthy controls.

Materials and methods: Ten PI-IBS patients and nine healthy controls participated. All PI-IBS patients met the Rome III diagnostic criteria for IBS and reported sustained symptoms at least 1 year after an episode of acute gastroenteritis. Intraepithelial lymphocytes and lamina propria lymphocytes (LPLs), isolated from mucosal tissue samples, were stained and analyzed for a comprehensive set of cell markers using flow cytometry.

Results: The number of LPLs in PI-IBS was significantly increased compared to those in healthy controls (p < 0.05). PI-IBS patients showed significantly increased proportions of CD45RO(+) CD4(+) activated/memory T cells (p < 0.05) and double-positive CD4(+) CD8(+) cells (p < 0.05), respectively, in the lamina propria. The number of CD19(+) LPLs was decreased in PI-IBS patients compared to healthy controls (p < 0.001).

Conclusion: This study presents new evidence that PI-IBS is associated with a sustained aberrant mucosal immune response and support future studies of anti-inflammatory or immune-modulating treatments in these patients.

BACKGROUND: The formation and accumulation of cholesterol crystals (CC) at the lesion site is a hallmark of atherosclerosis. Although studies have shown the importance of vascular smooth muscle cells (VSMCs) in the disease atherosclerosis, little is known about the molecular mechanism behind the uptake of CC in VSMCs and their role in modulating immune response.

METHODS: Human aortic smooth muscle cells were cultured and treated with CC. CC uptake and CC mediated signaling pathway and protein induction were studied using flow cytometry, confocal microscopy, western blot and Olink proteomics. Conditioned medium from CC treated VSMCs was used to study neutrophil adhesion, ROS production and phagocytosis. Neutrophil extracellular traps (NETs) formations were visualized using confocal microscopy.

RESULTS: VSMCs and macrophages were found around CC clefts in human carotid plaques. CC uptake in VSMCs are largely through micropinocytosis and phagocytosis via PI3K-AkT dependent pathway. The uptake of CC in VSMCs induce the release inflammatory proteins, including IL-33, an alarming cytokine. Conditioned medium from CC treated VSMCs can induce neutrophil adhesion, neutrophil reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. IL-33 neutralization in conditioned medium from CC treated VSMCs inhibited neutrophil ROS production and NETs formation.

CONCLUSION: We demonstrate that VSMCs due to its vicinity to CC clefts in human atherosclerotic lesion can modulate local immune response and we further reveal that the interaction between CC and VSMCs impart an inflammatory milieu in the atherosclerotic microenvironment by promoting IL-33 dependent neutrophil influx and NETs formation.

Urinary tract infections (UTIs) are among the most common infections in humans and are often caused by uropathogenic E. coli (UPEC). Trimethylamine N-oxide (TMAO) is a proinflammatory metabolite that has been linked to vascular inflammation, atherosclerosis, and chronic kidney disease. As of today, no studies have investigated the effects of TMAO on infectious diseases like UTIs. The aim of this study was to investigate whether TMAO can aggravate bacterial colonization and the release of inflammatory mediators from bladder epithelial cells during a UPEC infection. We found that TMAO aggravated the release of several key cytokines (IL-1β and IL-6) and chemokines (IL-8, CXCL1 and CXCL6) from bladder epithelial cells during a CFT073 infection. We also found that CFT073 and TMAO mediate increased release of IL-8 from bladder epithelial cells via ERK 1/2 signaling and not bacterial growth. Furthermore, we showed that TMAO enhances UPEC colonization of bladder epithelial cells. The data suggest that TMAO may also play a role in infectious diseases. Our results can be the basis of further research to investigate the link between diet, gut microbiota, and urinary tract infection.

Background and Aims: LDL-c is cleared from the circulation mainly by hepatic LDL-receptor mediated endocytosis. Defective LDL-c clearance and hence its elevation in circulation is one of the risk factors for myocardial infarction (MI). A soluble LDL-R (sLDL-R) exists in human plasma and exhibits strong correlation with circulating LDL-c and conditions that promote chronic inflammation. However, the mechanistic interplay between sLDL-R, inflammation and MI remains to be investigated.

Methods: In vitro studies using HepG2 cells treated with TNF-α, and a nested case-control study was conducted to investigate the relationship between plasma sLDL-R, TNF-α and risk of future MI.

Results: Stimulation of HepG2 cells with TNF-α induces release of sLDL-R with limited effect on surface expression of LDL-R. TNF-α induces gene expression of peptidases ADAM17 and MMP14 in HepG2 cells, and inhibition of ADAM17 and MMP-14 significantly reduces the TNF-α induced sLDL-R release. Although TNF-α treatment of HepG2 cells has limited effect on LDL-c endocytosis, HepG2 cells incubated with recombinant sLDL-R showed reduced LDL-c uptake in a dose-dependent manner. In a nested case-control study, baseline sLDL-R in plasma was positively correlated with plasma total cholesterol level. Further, a 2-fold increase in plasma sLDL-R was associated with 2.1x higher risk of future MI. Using mediation analyses, we determined that significant proportion of the association is mediated by elevation in plasma cholesterol level.

Conclusions: Our study suggests that sLDL-R is generated by TNF-α via membrane shedding. Further, an increase in sLDL-R could inhibit hepatic clearance of LDL-c increasing its half-life in the circulation and contributing to the pathogenesis of MI.