Umeå University's logo

Endometriosis, affecting 10% of women, is defined as implantation, survival, and growth of endometrium-like/endometriotic tissue outside the uterine cavity, causing inflammation, infertility, pain, and susceptibility to ovarian cancer. Despite extensive studies, its etiology and pathogenesis are poorly understood and largely unknown. The prevailing view is that the immune system of endometriosis patients fails to clear ectopically disseminated endometrium from retrograde menstruation. Exosomes are small extracellular vesicles that exhibit immunomodulatory properties. We studied the role of endometriotic tissue-secreted exosomes in the pathophysiology of endometriosis. Two exosome-mediated mechanisms known to impair the immune response were investigated: 1) downregulation of NKG2D-mediated cytotoxicity and 2) FasL- and TRAIL-induced apoptosis of activated immune cells. We showed that secreted endometriotic exosomes isolated from supernatants of short-term explant cultures carry the NKG2D ligands MICA/B and ULBP1-3 and the proapoptotic molecules FasL and TRAIL on their surface, i.e., signature molecules of exosome-mediated immune suppression. Acting as decoys, these exosomes downregulate the NKG2D receptor, impair NKG2D-mediated cytotoxicity, and induce apoptosis of activated PBMCs and Jurkat cells through the FasL- and TRAIL pathway. The secreted endometriotic exosomes create an immunosuppressive gradient at the ectopic site, forming a “protective shield” around the endometriotic lesions. This gradient guards the endometriotic lesions against clearance by a cytotoxic attack and creates immunologic privilege by induction of apoptosis in activated immune cells. Taken together, our results provide a plausible, exosome-based mechanistic explanation for the immune dysfunction and the compromised immune surveillance in endometriosis and contribute novel insights into the pathogenesis of this enigmatic disease.

Endometriosis, affecting 10% of women, is defined as implantation, survival, and growth of endometriumlike/endometriotic tissue outside the uterine cavity, causing inflammation, infertility, pain andsusceptibility to ovarian cancer. Despite extensive studies, its etiology and pathogenesis are poorlyunderstood and largely unknown. The prevailing view is that the immune system of endometriosispatients fails to clear ectopically disseminated endometrium from retrograde menstruation. Exosomes aresmall extracellular vesicles that exhibit immunomodulatory properties. We studied the role ofendometriotic tissue-secreted exosomes in the pathophysiology of endometriosis. Two exosome-mediatedmechanisms known to impair the immune response were investigated: 1) downregulation of NKG2Dmediatedcytotoxicity and 2) FasL- and TRAIL-induced apoptosis of activated immune cells. We showedthat secreted endometriotic exosomes isolated from supernatants of short-term explant cultures carry theNKG2D ligands MICA/B and ULBP1-3; and the proapoptotic molecules FasL and TRAIL on theirsurface, i.e. signature molecules of exosome-mediated immune suppression. Acting as decoys, theseexosomes downregulate the NKG2D receptor, impair NKG2D-mediated cytotoxicity and induce apoptosisof activated PBMC and Jurkat cells through the FasL- and TRAIL pathway. The secreted endometrioticexosomes create an immunosuppressive gradient at the ectopic site, forming a “protective shield” aroundthe endometriotic lesions. This gradient guards the endometriotic lesions against clearance by a cytotoxicattack and creates immunologic privilege by induction of apoptosis in activated immune cells. Takentogether, our results provide a plausible, exosome-based mechanistic explanation for the immunedysfunction and the compromised immune surveillance in endometriosis and contribute with novelinsights into the pathogenesis of this enigmatic disease.

Endometriosis is defined as the presence of endometrial-like tissue outside the uterine cavity. It has been suggested that the aberrant immunological mechanisms that cause dysfunction of immune cells and mediators are involved in the pathogenesis of endometriosis. There is substantial evidence of downregulated NK cell cytotoxicity and changes in inflammatory mediators such as cytokines in endometriosis. This research aimed to elucidate the immunosuppressive mechanisms in endometriosis, focusing on NK cells, the role of cytokines, and exosomes derived from endometriotic tissue.

Cytokines are small peptides/proteins used for intercellular communication, and regulate immune-effector functions in health and disease. In Paper I, real-time RT-qPCR and a set of primers and probes for 11 cytokines were used defining cytotoxic Th1, humoral Th2, regulatory Tr1/Th3, and inflammatory cytokine profiles. Cytokine mRNA expression in endometriotic tissue was compared with endometrium, and systemically with peripheral blood mononuclear cells (PBMC) from women with endometriosis and healthy controls. In addition, immunohistochemical staining with monoclonal antibodies was performed to investigate T-regulatory cells in endometriotic lesions. A downregulation of mRNA for cytokines that mediate cytotoxicity and antibody response was found in the endometriotic lesions. At the same time, there was an upregulation of inflammatory and T-regulatory cytokines in the endometriotic lesions, suggesting enhanced local inflammation and priming of an adaptive regulatory response. Consistent with these findings, T-­regulatory cells were abundant in the endometriotic lesions. These findings suggest that the ectopic implantation seen in endometriosis may be a consequence of increased inflammation and priming of adaptive T regulatory cells, resulting in impaired cytotoxicity and enhanced immune suppression. 

Exosomes are nanometer-sized extracellular vesicles of endosomal origin; they are produced by most cells in the body, convey intercellular communication and participate in both normal and pathological processes. Paper II show that endometriotic lesions produce high amounts of exosomes. The exosomes expressed on their surfaces the NKG2D ligands MICA/B and ULBP1-3 and the proapoptotic molecules FasL and TRAIL. These molecules are known as immunosuppressive signatures. Functional experiments were performed to show that these exosomes can downregulate the main activating NK receptor NKG2D on CTL and NK cells, reduce the killing ability of PBMC from healthy donors, and induce apoptosis of activated lymphocytes through the FasL/Fas pathway. The production and secretion of exosomes from the endometriotic tissue may be further enhanced by the vigorous local inflammation at ectopic sites. The results show that endometriotic lesions secrete immunosuppressive exosomes that inhibit cytotoxicity and promote apoptosis of activated immune cells. The exosomes form a “protective shield” around the endometriotic tissue thus promoting their survival.

NK cells are cytotoxic cells of the innate immune system. Human NK cells can be divided into two subsets: CD56^+bright and CD56^+dim. The CD^56+dim subset is more naturally cytotoxic, whereas the CD56^+bright subset produces more cytokines, but has low natural cytotoxicity. The majority (>90%) of circulating NK cells are CD56^+dim, whereas very few (0-10 %) are CD56^+bright. In Paper III a higher amount of CD56^+bright cells in serum was observed in one third of endometriosis patients compared to healthy controls. The amount of these cells was normalized after treatment with surgery, with or without medical treatment. Untreated patients had a lower expression of NKG2D receptors on their NK cells and CTLs compared to treated patients and healthy controls, which could be due to endometriotic exosomes carrying the NKG2D ligands that downregulate the receptor. Thus, surgery might have a beneficial effect on cytotoxic NK-cell function in endometriosis.

Endometriosis is considered a benign disease; however it has many features in common with tumors, and shares multiple microenvironmental hallmarks with cancer, including angiogenesis, immune dysregulation, inflammation, invasion, and metastasis. Paper II shows that endometriotic tissue secretes immunosuppressive exosomes. In Paper IV, exosomes in the peripheral blood of epithelial ovarian cancer (EOC) patients, and the impairment of the NKG2D receptor-ligand system in vivo before and after surgery, were studied. The serum exosomes isolated from the EOC patients carried the NKG2D ligands MICA/B and ULBP1-3. In functional experiments, the EOC exosomes downregulated the expression of the NKG2D receptor, and subdued NKG2D-­mediated cytotoxicity in NK cells from healthy donors in a similar manner to the endometriotic exosomes studied in Paper II. In Paper IV, surgery of the primary EOC tumor had a beneficial effect, alleviating the exosome-mediated suppression of NKG2D-mediated cytotoxicity. Thus, exosome-mediated immunosuppression is revealed as a common mechanism of action for immune escape in endometriosis and cancer. 

The results presented in this thesis provide novel and important insights into the function of the immune system in endometriosis, and give new explanations for why ectopic endometrial tissue persists and proliferates outside the uterine cavity. Furthermore, the immunosuppression in the microenvironment of endometriosis, which has many similarities with the local tumor microenvironment (TME), was investigated with a focus on the role of endometriotic exosomes. Taken together, this thesis contributes to understanding of the pathogenesis of endometriosis, and might be useful in identifying biomarkers for endometriosis and developing new immuno­modulatory therapies.

Endometrios är en sjukdom där vävnad som liknar livmoderslemhinna sitter på andra platser i kroppen än inuti livmodern. Man tror att endometrios beror på att livmoderslemhinna i mensblodet kommer ut i buken via äggledarna. Orsaken till att endometriosvävnaden blir kvar beror på ett otillräckligt immunsvar där immuncellerna inte fungerar som de ska. Det är känt att de så kallade ”mördarcellerna” NK cellerna (natural killer cells) har en nedsatt funktion vid endometrios och att de därför inte kan städa bort endometrioshärdarna. Det sker också en ökad inflammation i endometrioshärdarna som påverkar immunsvaret och förändrar utsöndringen av de molekyler som styr immunförsvaret. Syftet med denna avhandling är att belysa de immun­hämmande mekanismer som finns i endometrioshärdarna med fokus på NK celler, cytokiner samt de exosomer som produceras i endometrioshärdarna.

Cytokiner är små proteiner (äggviteämnen) som används för kommunikation mellan celler. De reglerar immunsystemet både hos friska individer och vid sjukdomar. I delarbete I undersöktes uttrycket av messengerRNA (mRNA) för olika cytokiner. mRNA är en molekyl som fungerar som en slags karta som beskriver hur proteiner ska byggas. Nivån av mRNA speglar hur mycket protein som sedan tillverkas. I avhandlingen undersöktes mRNA uttrycket för 11 cytokiner för att kunna skilja på ett cytotoxisk svar (avdödande), antikropps svar, regulatoriskt (immunhämmande) svar och inflammatoriskt svar. Ut­trycken av mRNA undersöktes i endometriosvävnad, livmoderslemhinna och blod och jämfördes med livmoderslemhinna och blod från friska kontroller. I endometriosvävnaden sågs en nedreglering av de cytokiner som förmedlar cytotoxicitet, samtidigt var både inflammatoriska och regulatoriska cytokiner uppreglerade, vilket kan betyda att immunsystemet försöker motverka den kraftiga inflammationen med ett regulatoriskt svar som stimulerar bildandet av regulatoriska T celler. Mikroskopering av endometriosvävnaden visade att det fanns mycket rikligt med regulatoriska T celler i endometriosvävnaden. Regulatoriska T celler är immunhämmande och hämmar den avdödande aktiviteten hos bl a NK celler vilket till viss del kan förklara NK cellernas nedsatta förmågan att döda vid endometrios.

Exosomer är mycket små vesikler (blåsor) som utsöndras från de flesta celltyper i kroppen. De fungerar som en slags “flaskpost” där de fraktar information både på ytan (flaskan) och inuti exosomen (brevet i flaskan). Med hjälp av exosomer så kan celler kommunicera med varandra utan direkt kontakt mellan cellerna. Exosomer är en del av både normala och sjukliga tillstånd. Delarbete II visade att endometriosvävnad tillverkar stora mängder exosomer. På sin yta bar dessa exosomer signalmolekyler som binder till den viktigaste aktiverande mottagarmolekylen (receptorn) på NK cellerna. För att en NK cell ska kunna döda andra celler så måste den aktiveras. Funktionella tester visade att exosomerna från endometriosvävnad kan nedreglera den aktiverande NK cells receptorn och på så sätt minska den avdödande aktiviteten hos immunceller i blodet från friska individer. Exosomerna utryckte även en sorts ”dödsmolekyler” på sin yta som kan framkalla ett inbyggt självmordsprogram (apoptos) hos verksamma immunceller. I funktionella tester framkallade exosomerna programmerat självmord hos immunceller i blodet från friska individer via den så kallade ”dödsreceptorn” på verksamma immunceller. Resultaten visar därmed att endometrios utsöndrar immunhämmande exosomer som förhindrar avdödning av sjukliga celler och främjar programmerat självmord av aktiva immunceller. Exosomerna fungerar som ”lockbeten” och formar en ”skyddande sköld” runt endometrioshärdarna så att de tillåts fortsätta att växa.

NK celler hos människor kan delas upp i två underkategorier: CD56^+bright och CD56^+dim. CD56^+dim celler är mer naturligt avdödande medan CD56^+bright celler tillverkar mer cytokiner och har låg naturlig förmåga att avdöda. Majoriteten (>90%) av NK celler i blod är CD56^+dim medan väldigt få NK celler (0-10%) är CD56^+bright. I delarbete III observerades ett högre antal CD56^+bright celler i blod hos drygt en tredjedel av endometrios patienternajämfört med friska kontroller. Denna förhöjning av antalet CD56^+bright celler i blod sänktes och normaliserades efter att patienterna opererat bort endo­metrioshärdarna med eller utan tillägg av hormonell behandling. Helt obehandlade endometrios patienter hade också ett lägre uttryck av den aktiverande NK receptorn jämfört med patienter behandlade med kirurgi och hormonell behandling. Detta skulle kunna bero på att exosomer från endometrioshärdarna hos de obehandlade patienterna nedreglerar receptorn.

Endometrios är en “godartad” (inte dödlig) sjukdom men har många kännetecken gemensamt med cancer såsom nybildandet av blodkärl, avvikande immunfunktion, inflammation, inträngande växt i vävnader och spridning. Delarbete II visar att endometrios utsöndrar immunhämmande exosomer. I delarbete IV undersöktes exosomer i blod vid äggstockscancer och hur de exosomerna hämmade aktiveringen av NK celler in vivo (på plats i kroppen) innan och efter det att cancern opererats bort. Exosomerna i blodet från cancerpatienterna bar på sin yta samma molekyler, som binder till den aktiverande NK receptorn, som de exosomer som studerades i delarbete II. Funktionella experiment visade att cancer exosomerna nedreglerade den aktiverande receptorn på NK cellerna och minskade den avdödande förmågan hos NK celler från friska kontroller på samma sätt som endometrios exosomerna i delarbete II. Delarbete IV visade också att det var gynnsamt att operera bort tumörvävnaden då det lindrade funktionsstörningen hos NK cellerna som orsakats av de immunhämmande exosomerna. Således visar sig immunhämmande exosomer vara en gemensam mekanism som både endometrios och cancer använder för att undkomma immunsystemet. 

Resultaten från denna avhandling ger nya och viktiga insikter om hur immunsystemet fungerar vid endometrios och ger oss nya förklaringar till varför endometriosvävnaden kan finnas kvar och tillväxa utanför livmodern. Sammanfattningsvis bidrar denna avhandling till förståelsen av sjukdoms­uppkomsten vid endometrios. Resultaten kan vara användbara för att finna nya markörer i tex blod för att diagnostisera/följa endometrios och utveckla nya behandlingar. 

Problem: Tumors compromise the patients’ immune system to promote their own survival. We have previously reported that HGSC exosomes play a central role, downregulating NKG2D cytotoxicity. Primary surgery's effect on tumor exosomes and NKG2D cytotoxicity in HGSC patients has not been studied before. The overall objective of this study was to explore the effect of surgery on the exosome-induced impairment of NKG2D cytotoxicity in HGSC.

Method of study: Paired pre- and post-operative blood samples were subjected to cell and exosome analyses regarding the NKG2D receptor and ligands, and NKG2D-mediated cytotoxicity. Lymphocytes were phenotyped by immunoflow cytometry. Exosomes, isolated by ultracentrifugation, and characterized by nanoparticle tracking analysis, transmission and immune electron microscopy and western blot were used in functional cytotoxic experiments. HGSC explant culture-derived exosomes, previously studied by us, were used for comparison.

Results: HGSC exosomes from patients’ sera downregulated NKG2D-mediated cytotoxicity in NK cells of healthy donors. In a subgroup of subjects, NKG2D expression on CTLs and NK cells was upregulated after surgery, correlating to a decrease in the concentration of exosomes in postoperative sera. An overall significantly improved NKG2D-mediated cytotoxic response of the HGSC patients’ own NK cells in postoperative compared to preoperative samples was noted.

Conclusions: Surgical removal of the primary tumor has a beneficial effect, relieving the exosome-mediated suppression of NKG2D cytotoxicity in HGSC patients, thus boostering their ability to combat cancer.

Problem: Endometriosis is a disease characterized by ectopic implantation of endometrium and impaired immune responses. To explore its pathogenic mechanisms, we studied the local and systemic cytokine mRNA profiles and their role in the immunity of patients with endometriosis and healthy controls.

Method of Study: mRNA for eleven cytokines defining cytotoxic Th1, humoral Th2, regulatory Tr1/Th3, and inflammatory cytokine profiles was characterized locally in endometriotic tissue and endometrium, and systemically in PBMCs from women with endometriosis and healthy controls, using real‐time qRT‐PCR. In addition, immunohistochemical stainings with monoclonal antibodies were performed looking for T regulatory cells in endometriotic lesions.

Results: We found a downregulation of mRNA for cytokines mediating cytotoxicity and antibody response and an upregulation of inflammatory and T‐regulatory cytokines in the endometriotic tissues and endometrium from the patients with endometriosis, suggesting enhanced local inflammation and priming of an adaptive regulatory response. Consistent with those findings, there was an abundancy of T regulatory cells in the endometriotic lesions.

Conclusions: The ectopic implantation seen in endometriosis could be possible as a consequence of increased inflammation and priming of adaptive T regulatory cells, resulting in impaired cytotoxicity and enhanced immune suppression.

Problem: Pre-eclampsia (PE), a severe human pregnancy disorder, is associated with exaggerated systemic inflammation, enhanced cytokine production, and increased shedding of microvesicles leading to endothelial dysfunction, coagulopathy, and extensive placenta destruction. The cause of PE is still unclear. Evidence suggests that its origin lies in the placenta and that the maternal immune system is involved. A shift in cytokine production in PE pregnancy promotes NK cell activation, suggested to be important in PE pathogenesis. In line with this suggestion, we studied NK cell cytotoxicity in peripheral blood of PE patients and controls and the mRNA expression of cytokines and of the NKG2D receptor and its ligands MICA/B and ULBP1-3 in PE- and normal placenta.

Method of study: The cytotoxic capacity of peripheral blood NK cells was analyzed using K562 target cells. The cytokine mRNA profiles and the mRNA expression of the NKG2D receptor and its ligands MICA/B and ULBP 1-3 in PE placenta were assessed and compared to those in normal placenta using real-time quantitative RT-PCR.

Results: The cytotoxicity of peripheral blood NK cells was upregulated in PE cases. Further, we found an enhanced inflammatory cytokine mRNA response combined with a dysregulated regulatory response and a significant mRNA overexpression of NKG2D receptor and its ligands MICA/B and ULBP in PE placenta.

Conclusion: The destruction of chorionic villi observed in PE placenta might be conveyed by an enhanced local cytotoxic response through the NKG2D receptor-ligand pathway, which in turn might be promoted by an intense inflammatory response not counteracted by regulatory cytokine responses.

Problem: Endometriosis is characterized by ectopic implantation of endometrial-like tissue and impaired immuneresponses such as the cytotoxic function of NK cells. NK cells can be divided into two subpopulations where theCD56^+bright cells produce more cytokines and have low natural cytotoxicity compared to CD56^+dim cells. Themajority (>90%) of circulating NK cells are CD56+dim whereas very few (0-10 %) are CD56+bright.

Method of Study: Using flow cytometry, NK cell subpopulations were analyzed in peripheral blood from 21individuals with endometriosis and 12 healthy controls. Furthermore, the NKG2D receptor expression on PBMCswas analyzed in untreated and treated endometriosis patients and controls.

Results: We found an increased level of CD56^+bright cells in 8 of 21 endometriosis patients. After surgery andhormonal treatment, the levels were normalized to that of controls. In a new cohort, the NKG2D receptorexpression on PBMCs was analyzed, with a lower expression in untreated patients compared to controls andpatients treated by surgery and hormones.

Conclusions: Our findings of a dominant CD56^+bright NK cell subpopulation in peripheral blood, anddownregulated levels of the NKG2D receptor on PBMCs, may explain the impaired cytotoxic immune functioncausing the persistence of ectopic endometrium in untreated endometriosis patients.