Background
Chronic pelvic pain (CPP) is a common symptom of endometriosis. Women with endometriosis are also at a high risk of suffering from anxiety, depression, and other psychological disorders. Recent studies indicate that endometriosis can affect the central nervous system (CNS). Changes in the functional activity of neurons, functional magnetic resonance imaging signals, and gene expression have been reported in the brains of rat and mouse models of endometriosis. The majority of the studies thus far have focused on neuronal changes, whereas changes in the glial cells in different brain regions have not been studied.
Methods
Endometriosis was induced in female mice (45-day-old; n = 6–11/timepoint) by syngeneic transfer of donor uterine tissue into the peritoneal cavity of recipient animals. Brains, spines, and endometriotic lesions were collected for analysis at 4, 8, 16, and 32 days post-induction. Sham surgery mice were used as controls (n = 6/timepoint). The pain was assessed using behavioral tests. Using immunohistochemistry for microglia marker ionized calcium-binding adapter molecule-1 (IBA1) and machine learning “Weka trainable segmentation” plugin in Fiji, we evaluated the morphological changes in microglia in different brain regions. Changes in glial fibrillary acidic protein (GFAP) for astrocytes, tumor necrosis factor (TNF), and interleukin-6 (IL6) were also evaluated.
Results
We observed an increase in microglial soma size in the cortex, hippocampus, thalamus, and hypothalamus of mice with endometriosis compared to sham controls on days 8, 16, and 32. The percentage of IBA1 and GFAP-positive area was increased in the cortex, hippocampus, thalamus, and hypothalamus in mice with endometriosis compared to sham controls on day 16. The number of microglia and astrocytes did not differ between endometriosis and sham control groups. We observed increased TNF and IL6 expression when expression levels from all brain regions were combined. Mice with endometriosis displayed reduced burrowing behavior and hyperalgesia in the abdomen and hind-paw.
Conclusion
We believe this is the first report of central nervous system-wide glial activation in a mouse model of endometriosis. These results have significant implications for understanding chronic pain associated with endometriosis and other issues such as anxiety and depression in women with endometriosis.
Graphical Abstract
In utero hematopoietic cellular transplantation (IUHCT) has the potential to treat congenital benign cellular disease without chemotherapy or radiation. Prenatal tolerance is essential in this regard. Previous studies involving prenatally created chimeras have shown that immunologically tolerant NK cells (friendly, fNK) from engrafter mice suppress allo Tc1 responses, while intolerant NK cells (hostile, hNK) from rejecter mice enhance allo Tc1 responses. Given that Th17 cells are also involved in allograft rejection, we sought to determine if rejecter hNK cells induce/expand allo-specific Th17/Tc17 cells.
We examined the helper potential of prenatally educated hNK cells in non-tolerant prenatal Balb/c --- > B6.Thy1.2 rejecter chimeras. Naïve Thy1.1+ responder T cells and allogenic target cells were adoptively transferred into rejecter or naïve hosts that had been depleted of endogenous T cells and fNK cells. We found increased Th17/Tc17 alloimmunity in the responder Thy1.1+T cell population in rejecters when compared to naïve hosts. Additionally, through ELISPOT, we confirmed increased IL-17A production by responder T cells when co-cultured with allo-specific hNK cells from rejecter mice. Hostile NK cells from rejecter mice produce higher levels of Th17 driving cytokines, TGF-beta 1, IL-6 and GM-CSF when compared to naïve hNK.
From these results, we conclude that prenatal rejecter hNK cells have an enhanced helper effect on Th17/Tc17 alloimmunity in vivo and in vitro. These findings support an evolving paradigm that prenatally educated NK cell can regulate the balance between Th1/Th17 and Treg functions.
Supported by NIH R01HL103745 Lurie Children’s Hospital Research Foundation (to A.F.S.)
Prenatal allospecific tolerance hinges on the developmental selection of NK cells expressing a friendly phenotype that is not cytotoxic to allogeneic donor cells. We wondered if prenatally educated friendly NK cells influenced allospecific T cell responses as an additional mechanism supporting prenatal tolerance. Therefore, we hypothesized that prenatally educated friendly NK cells exert suppressive effects on the induction of allospecific T cell responses.
To challenge this hypothesis, we examined the suppression potential of prenatally educated friendly NK cells through gain and loss of function experiments in stable prenatal Balb/c into B6.Thy1.2 chimeras (engrafter mice). We first depleted allospecific NK cells in host engrafter mice and then adoptively transferred Thy1.1+ naïve responder T cells along with CFSE-labeled allogenic or syngeneic target cells into the engrafter hosts. We found that the depletion of allospecific Ly49D+ NK cells in engrafter mice increased Thy1.1+ T cell alloimmunity in vivo. Conversely, adoptive transfer of friendly NK cells suppressed the induction of allospecific T cell responses in host mice. Furthermore, cultured friendly NK cells from engrafter mice showed stronger suppression on the induction of T cell alloimmunity in vitro than that of naïve mice. Lasltly, NK cell-mediated suppression of allospecific T cells required cell contact.
From these results, we conclude that prenatally educated friendly NK cell exert enhanced suppressive effects on T cell alloimmunity in vivo and in vitro. Depending on the context of their prenatal education, NK cells can differentially influence T cell responses in prenatal chimeras.
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