Cytokine Production In Tissue Research Articles

Perfused In Vitro Intestine Tissue Model to Evaluate the Role of Stromal and Immune Cells in Epithelial Response to Inflammatory Cues and Drug Therapies.

We establish an in vitro perfusion intestinal tissue bioreactor system tailored to study drug responses related to inflammatory bowel disease (IBD). The system includes key components including multiple human intestinal cell types (colonoids, myofibroblasts, and macrophages), a three-dimensional (3D) intestinal architecture, and fluid flow. Inclusion of myofibroblasts resulted in increased secretion of cytokines such as glypican-1 (GCP-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin 1-α (IL-1α), whereas inclusion of macrophages resulted in increased secretion of monocyte chemoattractant proteins (MCPs) demonstrating a significant role of both stromal and immune cell types in intestinal inflammation. The system is responsive to drug treatments, as reflected in the reduction of pro-inflammatory cytokine production in tissue in some treatment scenarios. While future studies are needed to evaluate more nuanced responses in an IBD context, the present study demonstrates the ability to establish a 3D intestinal model with multiple relevant cell types and flow that is responsive to both inflammatory cues and various drug treatment options.

The anti-inflammatory effects of Akkermansia muciniphila and its derivates in HFD/CCL4-induced murine model of liver injury

Inflammation plays a critical role in the promotion of hepatocyte damage and liver fibrosis. In recent years the protective role of Akkermansia muciniphila, a next-generation beneficial microbe, has been suggested for metabolic and inflammatory disorders. In this study, we aimed to evaluate the effects of live and pasteurized A. muciniphila and its extra cellular vesicles (EVs) on inflammatory markers involved in liver fibrosis in a mouse model of a high-fat diet (HFD)/carbon tetrachloride (CCl4)-induced liver injury. Firstly, the responses of hepatic stellate cells (HSCs) to live and pasteurized A. muciniphila and its EVs were examined in the quiescent and LPS-activated LX-2 cells. Next, the anti-inflammatory effects of different forms of A. muciniphila were examined in the mouse model of HFD/CCl4-induced liver injury. The gene expression of various inflammatory markers was evaluated in liver, colon, and white adipose tissues. The cytokine secretion in the liver and white adipose tissues was also measured by ELISA. The results showed that administration of live and pasteurized A. muciniphila and its EVs leads to amelioration in HSCs activation. Based on data obtained from the histopathological analysis, an improvement in gut health was observed through enhancing the epithelium and mucosal layer thickness and strengthening the intestinal integrity in all treatments. Moreover, live A. muciniphila and its EVs had inhibitory effects on liver inflammation and hepatocytes damage. In addition, the tissue cytokine production and inflammatory gene expression levels revealed that live A. muciniphila and its EVs had more pronounced anti-inflammatory effects on liver and adipose tissues. Furthermore, EVs had better effects on the modulation of gene expression related to TLRs, PPARs, and immune response in the liver. In conclusion, the present results showed that oral administration of A. muciniphila and its derivatives for four weeks could enhance the intestinal integrity and anti-inflammatory responses of the colon, adipose, and liver tissues and subsequently prevent liver injury in HFD/CCL4 mice.

Tissue eosinophilia correlates with mice susceptibility, granuloma formation, and damage during Toxocara canis infection.

AbstractAn increase in peripheral blood eosinophils in helminth infections is expected, and these cells are known to promote immunity against these parasites. However, studies have suggested that in some specific helminths, eosinophils may promote the needs and longevity of these parasites, and their role in these infections remains undefined, including in Toxocara canis infection. Thus, this study aimed to investigate the role of eosinophils in the context of larval migration of T. canis and the immunopathological aspects of infection. For this, we used wild-type mice and mice genetically deficient for the transcription factor GATA-binding factor 1 (GATA1−/−), infected with 1000 eggs of T. canis. At 0, 3, 14 and 63 days post-infection, parasite load, tissue cytokine production, leucocyte profile, bronchoalveolar lavage cells and histopathological analyses were carried out. Collectively, our results demonstrate that the presence of eosinophils mediates susceptibility to T. canis, inducing leucocytosis and the formation of granulomas, increasing the pulmonary and cerebral parasite load, and reducing the number of neutrophils, which may be necessary to control the infection.

COVID-19 transmission through host cell directed network of GPCR.

COVID-19 transmission through host cell directed network of GPCR.

Ex vivo culture of human atherosclerotic plaques: A model to study immune cells in atherogenesis

Background and aimsThe mechanisms that drive atherosclerotic plaque progression and destabilization in humans remain largely unknown. Laboratory models are needed to study these mechanisms under controlled conditions. The aim of this study was to establish a new ex vivo model of human atherosclerotic plaques that preserves the main cell types in plaques and the extracellular components in the context of native cytoarchitecture. MethodsAtherosclerotic plaques from carotid arteries of 28 patients undergoing carotid endarterectomy were dissected and cultured. At various time-points, samples were collected and analysed histologically. After enzymatic digestion, single cells were analysed with flow cytometry. Moreover, tissue cytokine production was evaluated. ResultsWe optimised the plaque dissection protocol by cutting plaques into circular segments that we cultured on collagen rafts at the medium–air interface, thus keeping them well oxygenated. With this technique, the relative presence of T and B lymphocytes did not change significantly during culture, and the sizes of lymphocyte subsets remained stable after day 4 of culture. Macrophages, smooth muscle cells, and fibroblasts with collagen fibres, as well as T and B lymphocyte subsets and CD16 natural killer cells, remained largely preserved for 19 days of culture, with a continuous production of inflammatory cytokines and chemokines. ConclusionsOur new model of ex vivo human atherosclerotic plaques, which preserves the main subsets of immune cells in the context of tissue cytoarchitecture, may be used to investigate important aspects of atherogenesis, in particular, the functions of immune cells under controlled laboratory conditions.

TH17 cells mediate inflammation in a novel model of spontaneous experimental autoimmune lacrimal keratoconjunctivitis with neural damage

Dry eye disease (DED) affects one third of the population worldwide. In prior studies, experimental autoimmune lacrimal keratoconjunctivitis (EALK) induced by desiccating stress in mice has been used as a model of DED. This model is complicated by a requirement for exogenous epithelial cell injury and administration of anticholinergic agents with broad immunologic effects. We sought to develop a novel mouse model of EALK and to demonstrate the responsible pathogenic mechanisms. CD4+CD45RBhigh naive T cells with and without CD4+CD45RBlow regulatory T cells were adoptively transferred to C57BL/10 recombination-activating gene 2 (Rag2)-/- mice. The eyes, draining lymph nodes, lacrimal glands, and surrounding tissues of mice with and without spontaneous keratoconjunctivitis were evaluated for histopathologic changes, cellular infiltration, and cytokine production in tissues and isolated cells. Furthermore, the integrity of the corneal nerves was evaluated using whole-tissue immunofluorescence imaging. Gene-deficient naive T cells or RAG2-deficient hosts were evaluated to assess the roles of IFN-γ, IL-17A, and IL-23 in disease pathogenesis. Finally, cytokine levels were determined in the tears of patients with DED. EALK developed spontaneously in C57BL/10 Rag2-/- mice after adoptive transfer of CD4+CD45RBhigh naive T cells and was characterized by infiltration of CD4+ T cells, macrophages, and neutrophils. In addition to lacrimal keratoconjunctivitis, mice had damage to the corneal nerve, which connects components of the lacrimal functional unit. Pathogenic T-cell differentiation was dependent on IL-23p40 and controlled by cotransferred CD4+CD45RBlow regulatory Tcells. TH17 rather than TH1 CD4+ cells were primarily responsible for EALK, even though levels of both IL-17 and IFN-γ were increased in inflammatory tissues, likely because of their ability to drive expression of CXC chemokines within the cornea and the subsequent influx of myeloid cells. Consistent with the findings of this model, the tears of patients with DED had increased levels of inflammatory cytokines, including IL-17A and TNF-α. We describe a novel model of spontaneous EALK that supports a role for TH17cells in disease pathogenesis and that will contribute to our understanding of autoimmune lacrimal keratoconjunctivitis in many human eye diseases, including DED.

Inulin-Type Fructans Modulates Pancreatic-Gut Innate Immune Responses and Gut Barrier Integrity during Experimental Acute Pancreatitis in a Chain Length-Dependent Manner.

Acute pancreatitis (AP) is a common abdominal inflammatory disorder and one of the leading causes of hospital admission for gastrointestinal disorders. No specific pharmacological or nutritional therapy is available but highly needed. Inulin-type fructans (ITFs) are capable of modifying gut immune and barrier homeostasis in a chemistry-dependent manner and hence potentially applicable for managing AP, but their efficacy in AP has not been demonstrated yet. The current study aimed to examine and compare modulatory effects of ITFs with different degrees of fermentability on pancreatic–gut immunity and barrier function during experimentally induced AP in mice. BALB/c mice were fed short (I)- or long (IV)-chain ITFs supplemented diets for up to 3 days before AP induction by caerulein. Attenuating effects on AP development were stronger with ITF IV than with ITF I. We found that long-chain ITF IV attenuated the severity of AP, as evidenced by reduced serum amylase levels, lipase levels, pancreatic myeloperoxidase activity, pancreatic edema, and histological examination demonstrating reduced pancreatic damage. Short-chain ITF I demonstrated only partial protective effects. Both ITF IV and ITF I modulated AP-associated systemic cytokine levels. ITF IV but not ITF I restored AP-associated intestinal barrier dysfunction by upregulating colonic tight junction modulatory proteins, antimicrobial peptides, and improved general colonic histology. Additionally, differential modulatory effects of ITF IV and ITF I were observed on pancreatic and gut immunity: ITF IV supplementation prevented innate immune cell infiltration in the pancreas and colon and tissue cytokine production. Similar effects were only observed in the gut with ITF I and not in the pancreas. Lastly, ITF IV but not ITF I downregulated AP-triggered upregulation of IL-1 receptor-associated kinase 4 (IRAK-4) and phosphor-c-Jun N-terminal kinase (p-JNK), and a net decrease of phosphor-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) p65 (p-NF-κB p65) nuclear translocation and activation in the pancreas. Our findings demonstrate a clear chain length-dependent effect of inulin on AP. The attenuating effects are caused by modulating effects of long-chain inulin on the pancreatic–gut immunity via the pancreatic IRAK-4/p-JNK/p-NF-κBp65 signaling pathway and on prevention of disruption of the gut barrier.

Kruppel-like factor 5 associates with melamine-cyanurate crystal-induced nephritis in rats

Melamine and cyanuric acid (M/CA), when orally administered together to rats, can induce crystal formation within renal tubules and cause acute kidney injury. To investigate the pathomechanism of crystal-induced nephritis, melamine and/or cyanuric acid were administered to 3-week-old (young) and 8-week-old (adult) rats, respectively. Crystal formation, blood urea nitrogen elevation, tubular cell injury and macrophage infiltration were noted in rats fed with M/CA, but not in rats fed with vehicle, melamine or CA alone. These parameters were significantly higher in young rats than those in adult rats fed with M/CA 200 mg/kg body weight (BW) for 3 days. Krüppel-like factor 5 (KLF5) was expressed on distal tubule cells, especially when crystals deposited within the lumens. Both mRNA and protein levels were higher in young rats than those in adult rats fed with M/CA (200 mg/kg BW). KLF5 expression has been shown to modulate renal tissue cytokine production, and we found that proinflammatory cytokines like monocyte chemoattractant protein-1 and interlukin-6 were increased in kidney tissues of young rats fed with M/CA for 3 days. In contrast, interlukin-10, an anti-inflammatory cytokine, was upregulated in kidneys of adult rats fed with M/CA for 3 days. Crystals are prone to deposition in distal tubules of young rats fed with M/CA. M/CA Crystal-related nephritis might be induced by the KLF5 expression, which modulated macrophage recruitment and proinflammatory cytokine production, subsequently leading to renal tubular injury and interstitial inflammation.

Leucine and calcitriol modulation of human airway inflammation and hyper‐reactivity

Obesity alters the balance of adipose tissue‐derived cytokines and increases airway inflammation and hyper‐reactivity. We previously found calcitriol and leucine to exert opposing effects on adipose tissue cytokine production, systemic inflammation, and monocyte adherence to vascular endothelial cells. We now propose that dietary calcium (via suppression of calcitriol) and leucine attenuate airway inflammation and reactivity by suppressing adipose tissue TNF‐α and enhancing adiponectin release. Human adipocytes were treated with calcitriol (10 nM) and leucine (0.5 mM) for 48 h and the conditioned medium (CM) were applied to human airway smooth muscle cells (HASMC). Calcitriol CM induced a 7‐fold increase in human leukocyte adhesion to HASMC (p<0.001) which was associated with increased TNF‐α receptor, VCAM and NFκB expression (~50%, p<0.02). In contrast, leucine CM decreased leukocyte adhesion (~50%, p<0.001) and NFκB (24%, p<0.02) and VCAM (21%, p<0.05) and increased adiponectin receptor 2 expression (29%, p<0.05). HASMC reactivity was measured as contractile responses to KCl and histamine under physiological flow and shear. Calcitriol CM augmented contractile responses to both agonists by 50 – 70% (p<0.001), while leucine inhibited the contractile response to KCl by 43% (p<0.001). These data suggest dietary calcium and leucine may suppress airway inflammation and reactivity.

Subcutaneous adipose tissue cytokine production is not responsible for the restoration of systemic inflammation markers during weight loss

It has been suggested that weight loss can improve systemic inflammation associated with obesity by decreasing the adipose production of pro-inflammatory cytokines. This suggestion, however, remains controversial. To analyse the effect of weight loss on peripheral inflammatory markers and subcutaneous adipocytokine production. Patients were studied at baseline, at the end of the weight loss period, and after 2 weeks of weight stabilisation. Nineteen morbid obese non-diabetic patients and 20 lean control subjects. During the weight loss period patients followed a 6-week low-calorie diet. Plasma levels of inflammatory markers, maximal in vitro whole-blood cytokine production, subcutaneous adipose tissue expression and content of several cytokines. Obese subjects had higher circulating levels of C reactive protein (CRP), serum amyloid A (SAA), interleukin IL-6, IL-1 and soluble tumor necrosis factor receptors (sTNFR). Weight loss was associated with a significant decrease in CRP, SAA, leucocytes and plasma IL-6. Maximal in vitro cytokine production of IL-1 and sTNFR1 increased during this period. Weight loss did not induce significant changes in the adipose concentrations of IL-6, IL-1 or sTNF-receptors. However, adipose expression of IL-6, IL-1, TNFalpha, membrane cofactor protein-1 and adiponectin increased at the end of the weight loss period. During weight maintenance, circulating inflammatory parameters increased and in some cases returned to baseline. A low-calorie diet is associated with an improvement in the systemic inflammatory status. This seems to be due to energy restriction rather than to adipose mass loss, since inflammatory levels return to baseline soon after weight stabilisation. Furthermore, a negative energy balance and fat mobilisation are associated with increased subcutaneous cytokine adipose expression.

Chronic Inflammation: Role of Adipose Tissue and Modulation by Weight Loss

Chronic inflammation has been linked with an increased risk of type 2 diabetes and cardiovascular disease. As an endocrine and inflammatory organ, adipose tissue is an important source of circulating pro-inflammatory cytokines. Current evidence strongly supports that chronic inflammation is associated with enlarged body fat mass. Moreover, inflammation is independently linked with abdominal, especially visceral fat mass, possibly due to the regional variation in adipose tissue cytokine production. In addition to pharmacological approaches, lifestyle modifications have been advocated for the treatment of chronic inflammation. A number of studies have indicated that either weight loss via energy restriction, or energy restriction plus other strategies (aerobic exercise, behavioral counseling, and liposuction), could reduce chronic inflammation. While the amount of weight loss tends to be important, exercise and other strategies may have additional effects. A few studies have reported weight loss effects on adipose tissue cytokine production. Weight loss reduces subcutaneous adipose tissue production of pro-inflammatory cytokines (i.e. interleukin 6, tumor necrosis factor alpha) and increases adipose expression of anti-inflammatory cytokines (i.e. interleukin 10, interleukin 1 receptor antagonist). More studies are needed to investigate the role of regional adipose tissue cytokine production in regulation of inflammation and the modulating effects of weight loss.

IgE(+), Kit(-), I-A/I-E(-) myeloid cells are the initial source of Il-4 after antigen challenge in a mouse model of allergic pulmonary inflammation.

IL-4 is generated within hours after antigen lung challenge and influences events that take place early in the induction of pulmonary inflammation. However, the cells responsible for this early IL-4 production in the lung are unknown. We sought to characterize the initial inflammatory events in the lung after antigen challenge and to identify cells responsible for producing IL-4 at early time points. Mice were sensitized with ovalbumin or passive IgE and challenged intranasally. Histologic measures of inflammation were used, and lung tissue cytokine production was analyzed by means of RT-PCR. Cells producing IL-4 were characterized by means of in situ hybridization and flow cytometry. IL-4 mRNA was detectable 100 minutes after challenge in sensitized animals. Blockade of this early IL-4 downregulated vascular cell adhesion molecule 1 mRNA expression and attenuated the early recruitment of eosinophils to the lung. CD4-depleted and mast cell-deficient mice both expressed early IL-4. Cellular analysis revealed the presence of IL-4 protein at 100 minutes exclusively in IgE(+) myeloid cells that did not express CD3, Kit, or I-A/I-E. Moreover, IL-4 production induced by means of passive IgE sensitization and abrogated in FcR gamma-chain-deficient mice supports the conclusion that this IL-4 production is dependent on IgE/gamma-chain interaction. IL-4 production by an IgE/gamma-chain-dependent mechanism occurs rapidly after allergen challenge. At these early time points, IL-4 is produced by a myeloid cell with the characteristics of a mouse basophil (IgE(+), Kit(-), I-A/I-E(-)). These data thus suggest that strategies targeting basophils should be considered in the treatment of early lung inflammation.

Expression of cytokines and immune mediators during chronic liver allograft rejection.

To determine the immune processes involved in chronic liver allograft rejection (CR) we examined in situ cytokine production in tissue from 15 patients with both clinical and histopathological diagnoses of CR. Total RNA was isolated from liver samples, reverse-transcribed and analyzed by RT-PCR for the production of proinflammatory cytokines and immunoregulatory mediators. Transcripts for the Th1-like cytokines IL-2 and IFN-gamma were detected in 53.3% and 46.7% of CR grafts, while they were detected in only 16% and 0% of stable grafts, respectively. The cytotoxic T cell mediator granzyme B was expressed in the majority of liver grafts undergoing CR, but was expressed only in a minority of stable grafts (80% vs. 16%, P < 0.05). The T cell product IL-5 was also significantly upregulated in CR as compared with stable livers (80% vs. 16%, P < 0.01). Other Th2 cytokines--IL-4 and IL-10--and macrophage products--IL-1 beta, IL-6, IL-8, TGF-beta, and TNF-alpha--were not substantially upregulated in CR grafts as compared with stable grafts. PDGF-beta transcripts were detected in the majority of the CR grafts, but were not detected in stable liver grafts (73% vs. 0, P < 0.05). By immunohistochemical staining, we observed that CD3+CD4+, and CD3+CD4- T cells were detected in CR grafts along with CD20+ B cells and CD68+ macrophages. There was, however, a predominant infiltration of CD3+CD4+ lymphocytes. Taken together, these data suggest that infiltrating cells produce proinflammatory and immunoregulatory cytokines that have a role in mediating graft damage in CR.

In situ hybridization of the mRNA for interferon-γ, interferon-αE, interferon-β, interleukin-1β and interleukin-6 and characterization of infiltrating cells in thyroid tissues

Cytokine mRNA production in the thyroid tissues of patients with various thyroid diseases was analysed by in situ hybridization. In addition, infiltrating leukocytes were characterized by immunhistologic studies using the alkaline phosphatase anti-alkaline phosphatase (APAAP) staining technique. The following clinical material was investigated: two cases of Graves' disease, one with high and the other with a low amount of infiltrating leukocytes as well as two cases of non-toxic goitre also showing considerable quantities of infiltrating cells. The hybridization was performed on tissue sections with antisense probes for interferon-gamma (IFN-γ), IFN-αE, IFN-β, interleukin-6 (IL-6) and IL-1β. A small number of individual cells were found to express high levels of mRNA for IFN-γ, IL-1β and measurable amounts of IL-6 throughout the tissue sections. However, IFN-αE or IFN-β were not detected. Cytokine expressing cells were noted in the tissue of one patient with Graves' disease and in two cases with non-toxic goitre. In these samples a high amount of infiltrating leukocytes (CD45 +) was detected, especially CD3 +, CD8 +, CD4 + and CD45RA + T cells, in addition to B cells and macrophages. In one case an unusually large amount of T cell receptor γ/δ + (TcR γ/δ +) cells was found. However, one sample of thyroid tissue derived from a patient with Graves' disease was poorly infiltrated and showed few cells expressing cytokines. In conclusion, using thyroid tissue as an example, our data suggest that the application of in situ hybridization with antisense RNA permits the study of cytokine production in tissues of both autoimmune and non-autoimmune origin.