Endotoxin Inflammatory Action on Cells by Dysregulated-Immunological-Barrier-Linked ROS-Apoptosis Mechanisms in Gut–Liver Axis

Our study highlighted the immune changes by pro-inflammatory biomarkers in the gut–liver-axis-linked ROS-cell death mechanisms in chronic and acute inflammations when gut cells are exposed to endotoxins in patients with hepatic cirrhosis or steatosis. In duodenal tissue samples, gut immune barrier dysfunction was analyzed by pro-inflammatory biomarker expressions, oxidative stress, and cell death by flow cytometry methods. A significant innate and adaptative immune system reaction was observed as result of persistent endotoxin action in gut cells in chronic inflammation tissue samples recovered from hepatic cirrhosis with the A-B child stage. Instead, in patients with C child stage of HC, the endotoxin tolerance was installed in cells, characterized by T lymphocyte silent activation and increased Th1 cytokines expression. Interesting mechanisms of ROS-cell death were observed in chronic and acute inflammation samples when gut cells were exposed to endotoxins and immune changes in the gut–liver axis. Late apoptosis represents the chronic response to injury induction by the gut immune barrier dysfunction, oxidative stress, and liver-dysregulated barrier. Meanwhile, necrosis represents an acute and severe reply to endotoxin action on gut cells when the immune system reacts to pro-inflammatory Th1 and Th2 cytokines releasing, offering protection against PAMPs/DAMPs by monocytes and T lymphocyte activation. Flow cytometric analysis of pro-inflammatory biomarkers linked to oxidative stress-cell death mechanisms shown in our study recommends laboratory techniques in diagnostic fields.


Introduction
In healthy conditions, the gut-liver axis assures organism-microbiota homeostasis [1,2].The gut barrier plays a vital role in host defense by the activating of enterocytes, goblet cells, Paneth cells, and immune cells [3,4].A group of sentinel goblet cells activates the NLRP6 inflammasome by ROS synthesis, and macrophages from intestinal lamina propria vasculature ensure defense against pathogens [5].The liver contains parenchymal cells and non-parenchymal cells, including liver sinusoidal endothelial cells, hepatic stellate cells, Kupffer cells, B cells, and T cells.Due to anatomical location and structure, LSEC is the first in contact with portal-delivered gut-derived pathogens, representing the hepatic barrier [6].
Bacterial translocation is bacteria and their metabolite's movement from the intestinal lumen to the portal bloodstream and mesenteric lymph nodes.BT increases in pathological conditions, determining a pro-inflammatory response [37,38].In cirrhotic animals, the dysregulated gut immune barrier determines increasing activated lymphocytes and IFN-γ and IL-17 production [37,39].The inflammation produced by dysbiosis mediated by monocytes and macrophages affects the gut barrier function by the TNF-α/TNFR1 signaling way [1].In cirrhosis patients, activated macrophages determine NO, IL-6, and IL-8 production under bacterial stimulation, affecting the gut barrier function [38].
The immune system and the hepatic barrier have role in the pathogenesis of ALD and NAFLD.Gut barrier dysfunction by increased intestinal permeability facilitates PAMPs portal influx, determining the hepatic inflammation [40].Progression of chronic liver diseases is associated with damages in gut defense, leading to a functional dysregulated gut barrier [27].
Our study highlighted the biomarkers pattern to evaluate the immunological dysfunction of the gut barrier.Immunological changes in gut barrier function were evaluated by pro-inflammatory biomarkers such as T helper lymphocytes (CD4), monocytes (CD14), and cytokines (IL-2, IL-6) by flow cytometry methods as the response to immune system activation.Cell death was studied using Annexin V-FITC/propidium iodide dual stain, and oxidative stress by total reactive oxygen species count using flow cytometry methods, to observe endotoxin tolerance installation due to gut immune and liver barrier dysfunctions, and changes in the apoptosis mechanism in gut cells determined by higher oxidative stress leading to pro-inflammatory and profibrogenic phenotypes.Our objective is to study the immune changes by pro-inflammatory biomarkers in gut-liver-axis-linked ROS-cell death mechanisms in chronic and acute inflammation when gut cells are exposed to endotoxins in patients with hepatic cirrhosis or steatosis.Gut immune barrier alterations in duodenal tissue samples by T lymphocytes and Th1 cytokines, and pro-inflammatory biomarkers using CD4 Alexa Fluor 488 and IL-2 PE stain, were used to establish their different patterns in liver cirrhosis or hepatic steatosis.Patients reported to controls are shown in Figures 1-3.

Pro-Inflammatory
Cell populations double positive (CD4+IL-2+) expressed using Alexa Fluor 488 and PE dual stain had an important role because they offered the possibility to observe changes in the gut immunological barrier function when presented with PAMPs/DAMPs or endotoxins action in cells, which may determine chronically or acute intestinal inflammation (CII or AII) patterns (Figures 1 and 2C).
Inflammation status by CD4+IL     Immune cell adaptation from the gut barrier by CD14 monocyte activation, and Th2 cytokines pro-inflammatory releasing, analyzed from different duodenal tissue samples recovered from patients with liver cirrhosis or hepatic steatosis reported to controls, were presented in Figures 4-6.

Cell Apoptosis in the Gut-Liver Axis
Apoptosis and necrosis, two major types of cell death, were studied using Annexin V-FITC/PI dual stain in different duodenal tissue samples recovered from patients with liver cirrhosis and hepatic steatosis reported to controls, as presented in Figures 7 and 8.
Changes in the gut immune barrier in hepatic steatosis patients determine a significant decrease of cell viability in CII than AII tissue samples reported to controls (CII: 44.29

Cell Apoptosis in the Gut-Liver Axis
Apoptosis and necrosis, two major types of cell death, were studied using Annexin V-FITC/PI dual stain in different duodenal tissue samples recovered from patients with liver cirrhosis and hepatic steatosis reported to controls, as presented in Figures 7 and 8  In hepatic steatosis patients, there were observed significantly higher values of cell apoptosis in CII than AII tissue samples (CII: LA: 33.92 ± 4.86 vs. AII: 0.00 ± 0.00, p < 0.01, CII: EA: 12.02 ± 7.19 vs. AII: 0.00 ± 0.00, p < 0.01, Figures 7A,D,G, and 8B,C).
Necrosis of gut cells presented significant differences between CII and AII tissue samples in patients with hepatic cirrhosis with A-B child (CII: 8. 51 7A,B,C,E,F, and 8D).

Oxidative Stress in the Gut-Liver Axis
In different duodenal cell samples recovered from patients with hepatic chronic diseases reported to controls.The oxidative cellular stress analyzed by total ROS is presented in Figure 9.

Oxidative Stress in the Gut-Liver Axis
In different duodenal cell samples recovered from patients with hepatic chronic diseases reported to controls.The oxidative cellular stress analyzed by total ROS is presented in Figure 9. ROS levels in hepatic steatosis patients showed significantly higher values in CII than in AII tissue samples and controls (CII: 66,266.66± 21,422.08 × 10 4 vs.AII: 12,751.25 ± 8594.51 × 10 4 , p < 0.05; vs. C: 371.51 ± 343.14 × 10 4 , p < 0.05; Figure 9A,E-G).
Figure 10A-L summarizes our main findings about immune changes in the gut-liveraxis-linked ROS-cell death mechanisms in chronic and acute inflammation when gut cells are exposed to endotoxins.Late apoptosis represents the chronic response to injury induction by gut immune barrier dysfunction, oxidative stress, and the liver-dysregulated barrier, being observed in duodenal tissue samples with chronic inflammation recovered from patients with hepatic cirrhosis and steatosis, compared with controls, using Annexin V-FITC/PI dual stain (Figure 10H,J).Late apoptosis represents the chronic response to injury induction by gut immune barrier dysfunction, oxidative stress, and the liver-dysregulated barrier, being observed in duodenal tissue samples with chronic inflammation recovered from patients with hepatic cirrhosis and steatosis, compared with controls, using Annexin V-FITC/PI dual stain (Figure 10H,J).
In the meantime, necrosis represents an acute and severe reply to endotoxin action in gut cells when the innate and adaptive functional immune system reacts to proinflammatory Th1 and Th2 cytokines releasing, offering protection against PAMPs/DAMPs by monocytes and T lymphocytes activation, being observed in duodenal tissue samples with acute inflammation recovered from patients with hepatic cirrhosis and steatosis, compared with controls, using Annexin V-FITC/PI dual stain (Figure 10G,I).
An interesting oxidative stress mechanism was observed in CII reported to AII tissue samples of hepatic chronic diseases, in gut cells, there is a constant action of endotoxins, which determines the installing of the endotoxin tolerance because of gut immune and liver barrier dysfunctions.Higher oxidative stress in gut cells determines changes in the apoptosis mechanism, leading to pro-inflammatory and profibrogenic phenotype status (Figure 10K,L).

Discussion
The gut barrier is a morphological functional mechanism that includes epithelial, immunological, vascular, and liver barriers [41].
Our study presents the adapted changes in the inflammatory potential of the gut immune environment highlighted by T lymphocytes, monocyte activation, and IL-2 production in patients with chronic hepatic diseases.The significant innate and adaptative immune system reaction resulting from constant endotoxin exposure of gut cells in chronic inflammation tissue samples recovered from hepatic cirrhosis with A-B child stage highlights the dysregulated gut immune barrier function.Instead, in patients with C child stage of HC, the endotoxin tolerance was installed in cells, characterized by T lymphocyte silent activation and increased Th1 cytokines expression.
Non-alcoholic fatty liver disease presents an increased risk of causing advanced liver diseases.Mechanisms of the intestinal barrier and permeability are disrupted in NAFLD.The gut barrier function is based on the microbiome integrity, mucus, enterocytes, immune cells, and vascular barrier [42][43][44][45].The immune barrier includes Paneth cells, and B and T lymphocytes [46].The epithelial barrier is the innate immunity site being formed by enterocytes producing defensins, goblet cells secreting mucus, tuft cells releasing IL-25 and IL-13, Paneth cells producing defensins, and M cells inducing secretory immunoglobulin A [47][48][49][50][51]. Innate immune cells are involved in metabolic homeostasis, releasing cytokines, and preceding the adaptive T lymphocytes response [52,53].Receptors with a role in PAMPs and DAMPs recognizing are implied in dendritic cell recruitment when the gut barrier is altered.Dendritic cells transport antigens useful to B and T lymphocyte maturation [53,54].The Kupffer cells have essential roles in liver barrier maintenance, phagocytizing the bacteria from the bloodstream, and eliminating the PAMPs and endotoxins.The Kupffer cells activation depends on human lipopolysaccharide-lipopolysaccharide-binding proteincomplex-stimulating myeloid cells by CD14 monocytes and Toll-like receptor 4 [55][56][57].
In liver cirrhosis, the presence of PAMPs and DAMPs associated with the altered gut barrier determines the transformation of tolerogenic properties of the liver into immunogenic and fibrinogenic properties based on the expansion of pro-inflammatory cells and cytokines from the extracellular matrix [68].The PAMPs binding to PRRs in tissues determines the immune cells' activation and pro-inflammatory cytokines' release.The presence of bacteria determines cell death when oxidative stress overwhelms the processing abilities of the endoplasmic reticulum, leading to unfolded protein responses determining the production of IL-6 and TNF pro-inflammatory cytokines.Positive feedback is assured by the IL-1, IL-6, IL-8, and TNF systemic cytokines using UFR activation in the liver, increasing systemic and hepatic inflammation [69][70][71].A persistent inflammation determines parenchymal and systemic immune cell apoptosis.In cirrhosis, CD14+CD16+ monocytes increase, expressing more TNF, determining a pro-inflammatory and profibrogenic phenotype [72,73].A recent study showed that in response to bacterial invasion, IL-2 pro-inflammatory cytokine secreted by immune cells affects T helper cells and decreases humoral immunity in advanced cirrhosis [74].
Hepatic stellate cell activation is responsible for NASH progression by TLR4 signaling way by increasing TNF-α expression [75,83], determining a release of pro-inflammatory cytokines and oxidative stress [84].In steatosis mice models, the presence of endotoxins triggers liver inflammation.Instead, in obese mice models, there were observed in portal circulation high levels of endotoxins and IL-1, IL-6, INF-γ, and TNF-α pro-inflammatory cytokines reported to control mice.HSCs were activated by enhanced sensitivity to LPS and increased cytokines levels, determining a dysregulated gut barrier [85].
In advanced cirrhosis, IL-2 pro-inflammatory cytokine is secreted by cells as a response to bacteria translocation [20,74].In patients with alcoholic hepatitis, there were observed increasing levels of TNF-α, IL-6, IL-8, and IL-18 pro-inflammatory cytokines.IL-6 protects against apoptosis [92], reducing alcoholic liver injury and inflammation [93], and has a protective effect in the ALD early phase [94].
Our study observed interesting ROS-cell death mechanisms in chronic and acute inflammation samples when gut cells are exposed to endotoxins and immune changes in the gut-liver axis.Late apoptosis represents the chronic response to injury induction by the gut immune barrier dysfunction, oxidative stress, and dysregulated liver barrier.Meanwhile, necrosis represents an acute and severe reply to endotoxin action on gut cells when the innate and adaptive functional immune system reacts to pro-inflammatory Th1 and Th2 cytokines releasing, offering protection against PAMPs/DAMPs by monocytes and T lymphocyte activation.An interesting oxidative stress mechanism was observed in chronic and acute inflammation samples of hepatic chronic diseases when there exists a constant action of endotoxins in gut cells, which determines the installation of endotoxin tolerance due to gut immune and liver barrier dysfunctions.Higher oxidative stress in gut cells determines changes in the apoptosis mechanism, leading to pro-inflammatory and profibrogenic phenotype status.
A significant source of reactive oxygen species in normal conditions is represented by mitochondria.TNF-α released from Kupffer cells stimulated by endotoxins leads to a decrease in mitochondrial complex III function [95].ROS is implied in the proinflammatory process.In the injured liver, pro-inflammatory cytokines and ROS produced by macrophages and infiltrating leukocytes determine HSCs transformation in activated phenotype, responsible for fibrosis, cirrhosis, and cancer development [96,97].Hepatic fibrosis is preceded by chronic inflammation.Liver inflammation is associated with necrosis and apoptosis of hepatocytes.In patients with liver cirrhosis, there were observed high levels of systemic IL-6 with a dysregulated acute phase response of the immune system [98].Another study reported that IL-1α and IL-6 serum levels were significantly increased in alcoholic cirrhosis patients, being correlated with the progression of liver cirrhosis [99].
Oxidative stress represents an essential aspect of research linked to intra-and extrahepatic disorders produced by NAFLD [100 -102].The oxidative stress mechanisms are linked to mitochondria and endoplasmic reticulum dysfunctions determining hepatic structure and function damages.These alterations of liver tissue by ROS affect extrahepatic tissues [103].In the liver, triglycerides and FFA induce lipotoxicity and oxidative stress, determining inflammation, mitochondrial dysfunction, apoptosis, and fibrosis.The progressive hepatocyte death by high oxidative stress promotes cirrhosis and HCC [104,105].In NAFLD, β-oxidation dysfunction determines the increasing of liver lipids levels and inflammation [106], leading to the pro-inflammatory and apoptotic response of the immune system [107,108].Activation of Kupffer cells by ROS triggers pro-inflammatory cytokines and chemokines released by macrophages [109,110].
In liver diseases, there are two significant types of cell death.Apoptosis is an early, chronic response to injury induction, whereas necrosis is an acute and severe reply.Biochemical mechanisms and morphological changes characterize apoptotic cells, including death receptor and mitochondria-dependent pathways [111][112][113].
Long-term use of alcohol leads to alcoholic liver disease development [114].Alcohol stimulates ROS production and leads to apoptosis via the oxidative stress mechanism.Alcohol's impact on hepatocytes includes endoplasmic reticulum stress and mitochondrial dysfunction [115].Alcohol alters endotoxin receptors, determining KCs' tolerance to endotoxins.Alcohol activates KCs to be sensitized by LPS via TLR4, promoting TNFα and ROS production [116].Pro-inflammatory factors determine liver dysfunction, apoptosis, necrosis, and fibrosis.The pro-inflammatory response of immune cells by increased ROS levels contributes to HSC activation [117].The activated HSCs' response to recurrent hepatic injury determines extra-cellular matrix protein accumulation, especially collagen type I. Apoptosis, inflammation, and fibrosis are characteristics of ADL [118].
NAFLD's pathological changes are related to dysregulated lipid metabolism and chronically pro-inflammatory-oxidative stress response [119].Macrophages and adipocytes secrete IL-6 and TNFα pro-inflammatory cytokines [120].ROS are neutralized by mitochondrial uncoupling protein two upregulation and limited synthesis of mitochondrial adenosine triphosphate [121].A lower antioxidant response induces sensitization of cells to mitochondrial and cellular apoptotic damages.FFAs activate the apoptosis pathway by Bim and Bax, determining mitochondrial permeabilization, cytochrome c release, and caspases activation [122,123].Pro-inflammatory cytokines from gut sources sensitize the liver to ROS and cellular lesions [124,125].
Interleukins assure the pro-inflammatory response of immune cells in their interaction with target cells [126].A higher systemic IL-6 level is a diagnostic biomarker to detect inflammatory conditions [127].Patients with acute and chronic liver diseases present increased IL-6 pro-inflammatory cytokines levels [128].In experimental obese mice models, IL-6 way signaling induced by TNFα lower level determined liver inflammation and carcinogenesis [129].In patients with chronic liver diseases, IL-6 levels in serum and liver tissue are biomarkers of disease progression [128,130].In liver cancer, IL-6 presents a protective role in fibrogenesis.Kupffer cells stimulate the releasing of pro-inflammatory cytokines, which are implied in tissue remodeling and fibrosis [131].Cytokines are implied in the regulation of inflammatory response and homeostasis [132].IL-6 has roles in cell differentiation and apoptosis blocking by modulating the specific gene transcription in hepatic inflammatory processes.IL-6 and transforming growth factor β induce IL-17 release from T lymphocytes.IL-2, IL-15, IL-18, and IL-21 pro-inflammatory cytokines stimulate IL-17 production from activated T lymphocytes [133].
The interactions between the liver and gut, mediated by the immune system, determine the liver transition from immune-tolerant to immune-active status, with TGF-β, IL-1, IL-6, and TNF-α pro-inflammatory cytokines production.A high oxidative stress resulting from the presence of endotoxin represents another mechanism for inducing liver damage and inflammation [150].
Going over the limitations of the study, such as a small number of patients or small pieces of duodenal biopsy, flow cytometric analysis of pro-inflammatory biomarkers, and apoptosis linked to oxidative stress mechanisms, may be used as a direction for future research in different maligned affections as efficient means of measurement in the diagnostic field.[151].In this paper, we studied only the immunological changes in the gut barrier by pro-inflammatory biomarkers linked to ROS-cell death mechanisms, not the entire gut barrier function in the gut-liver axis.The bacteria and endotoxins translocation from the liver to the gut and from the gut to the liver due to dysregulated hepatic barrier (patients with cirrhosis or steatosis) determine a pro-inflammatory and profibrogenic phenotype not only in the liver (already cirrhosis or steatosis presence) but also in gut cells being highlighted by ROS-cell apoptosis mechanisms.In function of immune response of patients determine an acute inflammation (when existing a functional immune response) or chronic inflammation (when gut cells present a modified phenotype in a pro-inflammatory and profibrogenic environment characterized by late apoptosis presence and high ROS levels).

Samples Selection
Duodenal biopsies (n = 116) were recovered from patients from the Gastroenterology Department, "Sf.Apostol Andrei" Clinical Emergency County Hospital, Constanta, Romania.Experimental groups were formed by patients with liver cirrhosis (n = 58) with acute or chronic intestinal inflammation (AII; CII) and patients with hepatic steatosis (n = 42) with acute or chronic intestinal inflammation (AII; CII).Experimental intestinal tissue samples were reported to control samples recovered from healthy patients.Tissue samples were homogenized with TissueRuptor II equipment (Qiagen, Hilden, Germany), being analyzed by flow cytometry methods (CD4 lymphocytes, CD14 monocytes, IL-2, IL-6 cytokines, cell apoptosis, and total reactive oxygen species) in The Cell Biology Department, CEDMOG, Ovidius University from Constanta, Romania.
In total, 5 µL of CD4 Alexa Fluor 488 and 5 µL of IL-2 PE were introduced in tubes with gut cells.An amount of 5 µL of CD14 Alexa Fluor 488 and 5 µL of IL-6 PE were added in other experimental tubes with cells.Control tubes with cells were realized for each experimental tube, and 5 µL of the mouse IgG1 negative control was added.All samples were vortexed and incubated for 25 min.at room temperature in darkness.In total, 1 mL of Flow Cytometry Buffer (Life Technologies, Europe BV, Bleiswijk, The Netherlands) was added in tubes.Glycoproteins and cytokines were identified by flow cytometry methods using the size and specificity of CD4, CD14, IL-2, and IL-6 expressions, and were analyzed with BL1 channel for Alexa Fluor 488 and BL2 channel for PE.

Cell Death Analysis
In total, 200 µL duodenal homogenized cells were introduced in flow cytometry tubes.An amount of 2 µL of Annexin V-FITC and 2 µL PI were added in tubes and kept in darkness for 30 min at room temperature.An amount of 1 mL of Flow Cytometry Buffer was added after incubation.Samples were analyzed by BL1 channel to 488 nm excitation, green emission for Annexin V-FITC, and BL2 channel in orange emission for propidium iodide.

Total ROS Activity Assay
In flow cytometry tubes, 50 µL of ROS Assay Stain 1x solution and 500 µL homogenized cell samples were mixed well.Samples were incubated at 37 • C in 5% CO 2 conditions for 60 min.Samples were analyzed by flow cytometry methods using the BL1 channel in green emission and 488 nm excitation for ROS.

Conclusions
Our study highlighted ROS-cell death mechanisms in chronic or acute inflammation when exposure of gut cells to endotoxins and immune changes in the gut-liver axis.
Late apoptosis is a chronic response to injury induction by gut immune barrier dysfunction, oxidative stress, and the liver-dysregulated barrier.In contrast, necrosis is an acute and severe reply to endotoxin action on gut cells when the innate and adaptive functional immune system reacts to pro-inflammatory Th1 and Th2 cytokines releasing, offering protection against PAMPs/DAMPs by monocytes and T lymphocytes activation in patients with hepatic cirrhosis and steatosis.
A persistent endotoxin action on gut cells determines an increase in oxidative stress, and endotoxin tolerance is installed due to gut immune and liver barrier dysfunctions.A higher oxidative stress in gut cells determines changes in the apoptosis mechanism, leading to pro-inflammatory and profibrogenic phenotype status.

Figure 3 .
Figure 3. Pro-inflammatory biomarkers.CD4 Helper T cells positive or negative populations expressed by Alexa Fluor 488 stain, Interleukine-2 (IL-2) positive or negative expressions by PE stain.Legend: (A,H) healthy patient C; (B-N) patients with hepatic cirrhosis (HC) with A child (B,E,I,L) or C child (C,F,J,M,J) stages by Child-Pugh score or hepatic steatosis (HS; D,G,K,N) with acute intestinal inflammation (B-D,I-K) or chronic intestinal inflammation (E-G,L-N).2.1.2.Monocytes and Th2 Cytokines Expressions in Acute or Chronically Intestinal Inflammation Tissue Samples Recovered from Liver Cirrhosis or Hepatic Steatosis Patients

Figure 3 .
Figure 3. Pro-inflammatory biomarkers.CD4 Helper T cells positive or negative populations expressed by Alexa Fluor 488 stain, Interleukine-2 (IL-2) positive or negative expressions by PE stain.Legend: (A,H) healthy patient C; (B-N) patients with hepatic cirrhosis (HC) with A child (B,E,I,L) or C child (C,F,J,M,J) stages by Child-Pugh score or hepatic steatosis (HS; D,G,K,N) with acute intestinal inflammation (B-D,I-K) or chronic intestinal inflammation (E-G,L-N).2.1.2.Monocytes and Th2 Cytokines Expressions in Acute or Chronically Intestinal Inflammation Tissue Samples Recovered from Liver Cirrhosis or Hepatic Steatosis PatientsImmune cell adaptation from the gut barrier by CD14 monocyte activation, and Th2 cytokines pro-inflammatory releasing, analyzed from different duodenal tissue samples recovered from patients with liver cirrhosis or hepatic steatosis reported to controls, were presented in Figures4-6.

Figure 6 .
Figure 6.Pro-inflammatory biomarkers.T lymphocytes (monocytes) positive or negative cell populations expressed using Alexa Fluor 488 stain; Interleukine-6 (IL-6) positive or negative expressions by PE stain.Legend: (A,H) healthy patient C; (B-N) patients with hepatic cirrhosis (HC) with A child (B,E,I,L) or C child (C,F,J,M,J) stages by Child-Pugh score or hepatic steatosis (HS; D,G,K,N) with acute intestinal inflammation (B-D,I-K) or chronic intestinal inflammation (E-G,L-N).