Menstruation is triggered by a fall in circulating progesterone (P4), and to a lesser extent, oestradiol (E2) concentrations, and characterised by classical inflammatory features in the endometrium: breakdown of the basal lamina, tissue oedema and an influx of migratory leucocytes. During and following menstruation, endometrial inflammation is resolved and the endometrium is repaired. The successful resolution of acute inflammation in other tissues involves apoptosis and the phagocytic clearance of apoptotic cells. Human endometrial tissues were collected with informed patient consent and local research ethics committee approval. C57Bl/6 mice underwent an induced menstruation protocol (via sequential E2 and P4 exposure followed by P4 withdrawal), both with and without experimental inhibition of apoptosis (using the pan-caspase inhibitor, Q-VD-OPh). Coordinated apoptosis and neutrophil recruitment were hypothesised to be components of the menstrual event and to precede menstrual shedding in the human endometrium. Immunoreactivity histoscoring for cleaved caspase-3 (CC3) revealed extensive apoptosis in the normal human endometrium early in the ‘perimenstrual’ period, and careful stereological delineation of neutrophil (elastase+) recruitment showed a significant influx coincident with menstrual tissue breakdown. Apoptosis and neutrophil recruitment were hypothesised to follow similar courses in the endometria of mice undergoing an induced menstruation protocol, recapitulating human menstrual events. Immunoreactivity histoscoring for CC3 and stereological investigation into neutrophil (Ly6G+) recruitment in mouse endometrial tissues revealed almost identical extents and timings of apoptosis and neutrophil recruitment in women. Whole genome array evidence of differential apoptosis-related gene transcription in the endometria of women with heavy menstrual bleeding (HMB) compared to those of women with normal menstrual bleeding (NMB) led to the hypothesis that apoptosis may be dysregulated in women with HMB and that perhaps this may delay timely repair of the endometrium and lead to prolonged bleeding in consequence. Candidate differentially-regulated gene transcripts identified by the whole genome array were validated by means of RT-qPCR, although immunoreactivity histoscoring for CC3 did not reveal any differences in apoptosis or its localisation between women with NMB and HMB at the menstrual cycle time-points examined. Building on evidence of apoptotic transcriptional dysregulation in the endometria of women with HMB, it was hypothesised that experimental inhibition of apoptosis (via Q-VD-OPh) in a mouse model of induced menstruation could delay endometrial repair and delay resolution of endometrial inflammation. Some evidence of delayed early repair was obtained, alongside the discoveries of delayed inflammatory gene transcription and increased decidual proliferation (BrdU+) in apoptosis-inhibited mice. Apoptosis precedes the classical inflammatory features of menstruation in the human and mouse endometrium, with inhibition of apoptosis in the latter altering repair and the inflammatory micro-environment. An apoptosis-inhibited mouse model of menstruation may therefore represent a viable model for the further study of heavy menstrual bleeding.

Cancer cachexia is a complex syndrome that can affect up to 80% of cancer patients. Among the symptoms involved in cancer cachexia progression, the establishment of a systemic inflammation and the imbalance in glucose metabolism homeostasis take an important part in this profile. The aim of the present study was to further evaluate the role of cancer-induced inflammation in the impairment of pancreatic beta cell function in solid Ehrlich carcinoma-bearing mice. For that, we have focused the study in the pro-inflammatory mechanisms involved on β-cell death. We have observed that tumor-bearing animals developed an aggressive pancreatic inflammatory status 14 days after tumor cells inoculation. The increase of pro-inflammatory cytokines followed by an up-regulation of important transcription factors such as NF-κB and STAT-1 and its related genes, reveled a similar outline for β-cell death found in type 1 diabetes. Furthermore, expression of pro-apoptotic Bcl-2 family members followed by caspases activation was increased in pancreatic islets of tumor-bearing animals and the expression of anti-apoptotic members was decreased. We have also observed an increase in β-cell death and ER stress components, as well as a decrease in insulin content cells together with an increase in alpha cells content. Overall, our results provide strong evidences that pancreatic β-cells in tumor-bearing animals are widely affected by tumor presence and systemic inflammation establishment. Interestingly, it was shown a similarity with mechanisms of β-cell death found in type 1 diabetes. Although the exactly mechanisms behind the changes found in carbohydrate metabolism in cancer cachexia is still unclear, our data can help to clarify, at least in part, this profile and would serve as a basis for development of new strategies to prevent cachexia progression and to improve the quality of life of cancer patients. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

Initiation and perpetuation of inflammatory bowel diseases (IBD) may result from an exaggerated mucosal immune response to the luminal microbiota in a susceptible host. We proposed that this may be caused either 1) by an abnormal microbial composition or 2) by weakening of the protective mucus layer due to excessive mucus degradation, which may lead to an easy access of luminal antigens to the host mucosa triggering inflammation. We tested whether the probiotic Enterococcus faecium NCIMB 10415 (NCIMB) is capable of reducing chronic gut inflammation by changing the existing gut microbiota composition and aimed to identify mechanisms that are involved in possible beneficial effects of the probiotic. To identify health-promoting mechanisms of the strain, we used interleukin (IL)-10 deficient mice that spontaneously develop gut inflammation and fed these mice a diet containing NCIMB (106 cells g-1) for 3, 8 and 24 weeks, respectively. Control mice were fed an identically composed diet but without the probiotic strain. No clear-cut differences between the animals were observed in pro-inflammatory cytokine gene expression and in intestinal microbiota composition after probiotic supplementation. However, we observed a low abundance of the mucin-degrading bacterium Akkermansia muciniphila in the mice that were fed NCIMB for 8 weeks. These low cell numbers were associated with significantly lower interferon gamma (IFN-γ) and IFN-γ-inducible protein (IP-10) mRNA levels as compared to the NCIMB-treated mice that were killed after 3 and 24 weeks of intervention. In conclusion, NCIMB was not capable of reducing gut inflammation in the IL-10-/- mouse model. To further identify the exact role of A. muciniphila and uncover a possible interaction between this bacterium, NCIMB and the host in relation to inflammation, we performed in vitro studies using HT-29 colon cancer cells. The HT-29 cells were treated with bacterial conditioned media obtained by growing either A. muciniphila (AM-CM) or NCIMB (NCIMB-CM) or both together (COMB-CM) in Dulbecco’s Modified Eagle Medium (DMEM) for 2 h at 37 °C followed by bacterial cell removal. HT-29 cells treated with COMB-CM displayed reduced cell viability after 18 h (p<0.01) and no viable cells were detected after 24 h of treatment, in contrast to the other groups or heated COMB-CM. Detection of activated caspase-3 in COMB-CM treated groups indicated that death of the HT-29 cells was brought about by apoptosis. It was concluded that either NCIMB or A. muciniphila produce a soluble and heat-sensitive factor during their concomitant presence that influences cell viability in an in vitro system. We currently hypothesize that this factor is a protein, which has not yet been identified. Based on the potential effect of A. muciniphila on inflammation (in vivo) and cell-viability (in vitro) in the presence of NCIMB, we investigated how the presence of A. muciniphila affects the severity of an intestinal Salmonella enterica Typhimurium (STm)-induced gut inflammation using gnotobiotic C3H mice with a background microbiota of eight bacterial species (SIHUMI, referred to as simplified human intestinal microbiota). Presence of A. muciniphila in STm-infected SIHUMI (SIHUMI-AS) mice caused significantly increased histopathology scores and elevated mRNA levels of IFN-γ, IP-10, tumor necrosis factor alpha (TNF-α), IL-12, IL-17 and IL-6 in cecal and colonic tissue. The number of mucin filled goblet cells was 2- to 3- fold lower in cecal tissue of SIHUMI-AS mice compared to SIHUMI mice associated with STm (SIHUMI-S) or A. muciniphila (SIHUMI-A) or SIHUMI mice. Reduced goblet cell numbers significantly correlated with increased IFN-γ (r2 = -0.86, ***P<0.001) in all infected mice. In addition, loss of cecal mucin sulphation was observed in SIHUMI-AS mice. Concomitant presence of A. muciniphila and STm resulted in a drastic change in microbiota composition of the SIHUMI consortium. The proportion of Bacteroides thetaiotaomicron in SIHUMI, SIHUMI-A and SIHUMI-S mice made up to 80-90% but was completely taken over by STm in SIHUMI-AS mice contributing 94% to total bacteria. These results suggest that A. muciniphila exacerbates STm-induced intestinal inflammation by its ability to disturb host mucus homeostasis. In conclusion, abnormal microbiota composition together with excessive mucus degradation contributes to severe intestinal inflammation in a susceptible host. / Die Initiation and die Manifestation von entzündlichen Darmerkrankungen (inflammatory bowel diseases - IBD) können aus einer übersteigerten mukosalen Immunreaktion auf die luminale Mikrobiota in einem empfänglichen Wirt resultieren. Wir schlagen vor, dass dies entweder durch 1) eine abnormale mikrobielle Zusammensetzung oder 2) die Abschwächung der schützenden Schleimschicht, eingeleitet durch deren fortgeschrittenen Abbau, verursacht werden kann. Diese Entwicklung ermöglicht einen erleichterten Zugang des luminalen Antigens zu der Mukosa des Wirts und somit die Auslösung der Entzündung. Wir haben getestet, ob das probiotische Bakterium Enterococcus faecium NCIMB 10415 (NCIMB) in der Lage ist, der chronischen Darmentzündung durch Veränderung der Zusammensetzung der Darmmikrobiota entgegenzuwirken und strebten an, die zugrunde liegenden Mechanismen der probiotischen Wirkungsweise zu identifizieren. Für die Aufklärung der gesundheitsfördernden Mechanismen dieses Bakterienstammes wurden Interleukin-10 defiziente Mäuse verwendet, die spontan eine Darmentzündung entwickeln. Den Mäusen wurde für 3, 8 und 24 Wochen eine NCIMB enthaltende Diät verabreicht. Nach der Fütterung waren keine eindeutigen Unterschiede zwischen den Gruppen hinsichtlich der Genexpression von pro-inflammatorischen Zytokinen und der Zusammensetzung der Darmmikrobiota zu beobachten, obwohl eine geringere Zellzahl des schleimabbauenden Bakteriums Akkermansia muciniphila in den mit NCIMB gefütterten Mäusen nach 8 Wochen festgestellt wurde. Daraus folgt, dass NCIMB nicht in der Lage ist, dem Verlauf der Darmentzündung im IL-10-/--Mausmodell entgegenzuwirken. In der nachfolgenden Studie wurde untersucht, wie die Anwesenheit von A. muciniphila den Ausprägungsgrad einer intestinalen Salmonella enterica Typhimurium (STm) induzierten Darmentzündung beeinflusst. Dafür wurden gnobiotische C3H-Mäuse mit einem mikrobiellen Hintergrund von acht Bakterienspezies (SIHUMI) verwendet. Die gleichzeitige Anwesenheit von A. muciniphila und STm verursachte eine drastische Veränderung der Mikrobiota-Zusammensetzung des SIHUMI-Konsortiums. Diese Ergebnisse zeigen, dass A. muciniphila durch seine Fähigkeit, die Homöostase/Selbstregulation der Schleimbildung zu stören, die STm-induzierte Darmentzündung verschärft. Es kann geschlußfolgert werden, dass eine abweichende Zusammensetzung der Mikrobiota in Kombination mit einem massiven Abbau des Mucus zur schweren intestinalen Entzündung im empfänglichen Wirt beiträgt.

Thesis (Ph.D. (Biochemistry)) -- University of Limpopo, 2021 / Introduction: Rift Valley fever virus (RVFV) is an arthropod-born RNA zoonotic virus causing Rift Valley fever (RVF) disease. RVFV is prevalent across sub-Saharan Africa and the Arabian Peninsula with no existing effective and approved antiviral remedies for humans or animals. RVFV has developed mechanisms to hide from immune recognition and induce anti-apoptosis processes to keep the infected host cells viable in an attempt to advance their viral progeny. RVFV is a single-stranded enveloped RNA genome virus composed of 3 segments; the L, M and S segments. The S segment is known to encode a non-structural protein (NSs) identified to be the main virulence factor promoting viral replication through immune suppression. RVFV elicits a set of diverse symptoms ranging from a febrile illness to more severe symptoms that usually culminate in life-threatening haemorrhagic fever with high fatality rates. Thus, this study was designed to investigate the efficacy of lithium as a potential drug for reduction of RVFV load and amelioration of imbalanced and dysregulated inflammatory responses observed in Huvec and Raw 264.7 macrophages infected with this virus. Methods and results: The MTT and Cyquant viability assays were used to demonstrate that lithium exerts no cytotoxic effects on non-infected Raw 264.7 macrophage cells but rather promotes cell growth and proliferation. Conversely, lithium was shown to significantly induce cell death in RVFV-infected Raw 264.7 macrophages. The Annexin-V/PI apoptosis assay was employed to demonstrate that RVFV induces apoptosis as a mode of cell death on Raw 264.7 cells. RVFV-induced apoptosis was accompanied by antagonistic Bax/Bcl-2 protein expression ratios. RVFV-infected cells treated with lithium resulted in higher levels of apoptosis signals compared to untreated RVFV-infected cells. Analysis of apoptosis stages using the real-time cell analyser (RTCA) also revealed that lithium induced early forms of apoptosis in RVFV-infected cells. Interestingly, induction of early apoptosis in these cells corresponds with lower viral load, probably as a result of early inhibition of viral progeny replication, as determined using viral titration assay. Immune response profiles elicited in Raw 264.7 macrophages infected with RVFV and treated with lithium were monitored. An ELISA assay was used to determine the effect of lithium on cytokines and chemokine production in this cell model. The results obtained showed that lithium significantly stimulated production of IFN-γ as RVFV-infected lithium-treated cells produced high levels of IFN-γ compared to lithium-free RVFV-infected control cells. Furthermore, in the same setting, the secondary pro-inflammatory cytokine, IL-6, and chemokine, RANTES, were stimulated by lithium 12 hrs post-infection (pi). Lithium was shown to significantly stimulate TNF-α production as early as 3 hrs pi. In addition to TNF-α expression, the expression of the regulatory cytokine, IL-10, was significantly stimulated by lithium with the highest expression peak at 12 hrs pi. As determined using the H2DCF-DA and DAF-2 DA florigenic assays, reduced production of the ROS and RNS was observed in RVFV-infected lithium-treated cells as opposed to untreated RVFV-infected controls. This was further supported by the Western blot assay results that showed low expression of the iNOS while upregulating expression of heme oxygenase and IκB in RVFV-infected lithium-treated cells. Results from immunocytochemistry and Western blot assays revealed that lithium inhibits NF-κB nuclear translocation in RVFV-infected cells compared to lithium-free RVFV-infected cells and 5 mg/ml LPS controls. This study hypothesises persistent and deregulated inflammation as the central phenomenon responsible for endothelial damage and haemorrhagic fever in RVFV pathogenesis. Supernatants were collected from RVFV-infected macrophage cells treated with lithium and their effects on the integrity of endothelial cells were evaluated. The xcelligence real-time cell analyser system (RTCA) and transwell assay that measure endothelial monolayer integrity were used to demonstrate that lithium protects endothelial cells from RVFV-induced cellular damage. Moreover, lithium was shown to upregulate expression of cytoplasmic molecules such as α and β-catenins involved in attaching the cadherin molecules to the actin cytoskeleton on the endothelial cell. Expression of α-catenins, talins, zyxins and vinculins that attach integrins to the extracellular matrix and to other cells were observed to be upregulated by supernatants from RVFV-infected Raw 264.7 macrophage cells treated with lithium. Endothelial cell monolayer exposed to supernatants from RVFV-infected lithium-treated Raw 264.7 cells displayed upregulated expression of transmembrane molecules such as E-cadherins and N-cadherins. However, expression of VE-cadherins was observed to be lower compared to those treated with supernatants from lithium-free RVFV-infected Raw 264.7 control cells. Conclusion: These findings propose that lithium limits viral replication and viral load in macrophages by inducing early apoptosis in RVFV-infected cells. Since lithium was shown to promote Raw 264.7 macrophage proliferation, it is thus suggested that the use of lithium as an RVFV antiviral drug is less likely to elicit leukocytopenia. Lithium seems to regulate excessive inflammation in RVFV-infected Raw 264.7 macrophages by modulating the NF-kB signalling pathway. The endothelial integrity observed in the permeability assays has been supported by the expression of the molecules involved in keeping the cell to cell adhesion intact. This study links endothelial integrity patterns exerted by lithium with lowered production of inflammatory mediators such as ROS and RNS as these molecules are involved in destabilisation of cell junctions. Results from this study point towards the use of lithium as a potential treatment for RVFV infections by limiting viral replication, restricting viral spread and restoring the inflammation-regulating machinery. Key words. Lithium, Rift Valley fever virus, NF-kB, endothelial integrity, inflammation and apoptosis / Poliomyelitis Research foundation and National Research Foundation