Aberrant CD8+T cells drive reproductive dysfunction in female mice with elevated IFN-γ levels.

Introduction: Interferon-gamma (IFN-γ) is pivotal in orchestrating immune responses during healthy pregnancy. However, its dysregulation, often due to autoimmunity, infections, or chronic inflammatory conditions, is implicated in adverse reproductive outcomes such as pregnancy failure or infertility. Additionally, the underlying immunological mechanisms remain elusive. Methods: Here, we explore the impact of systemic IFN-γ elevation on cytotoxic T cell responses in female reproduction utilizing a systemic lupus-prone mouse model with impaired IFN-γ degradation. Results: Our findings reveal that heightened IFN-γ levels triggered the infiltration of CD8+T cells in the pituitary gland and female reproductive tract (FRT), resulting in prolactin deficiency and subsequent infertility. Furthermore, we demonstrate that chronic IFN-γ elevation increases effector memory CD8+T cells in the murine ovary and uterus. Discussion: These insights broaden our understanding of the role of elevated IFN-γ in female reproductive dysfunction and suggest CD8+T cells as potential immunotherapeutic targets in female reproductive disorders associated with chronic systemic IFN-γ elevation.

Unusual Urothelial Tumors and Refractory Uremia Due to Balkan Endemic Nephropathy: A Case Report.

Balkan endemic nephropathy (BEN) is a rare progressive chronic renal disease found in residents living along the Balkan peninsula. We present a 92-year-old female who complained initially of cardio-respiratory symptoms and was found to have an acute hypoxemic respiratory failure with hypervolemia. The patient underwent computed tomography imaging and was found to have bilateral pleural effusions and moderate left-sided renal atrophy with left-sided hydronephrosis. The patient underwent diuresis for fluid overload and was treated with broad-spectrum antibiotics for hospital-acquired pneumonia. Further urological work-up revealed masses in the posterior bladder wall and left ureteropelvic junction. A biopsy of the posterior bladder wall mass confirmed high-grade papillary urothelial carcinoma. A review of the epidemiological history revealed the patient lived in Kosovo/former Yugoslavia for several decades following birth. A review of old records revealed the patient had chronic kidney disease (CKD) that was not fully explained by other causes, such as hypertension or diabetes. Given the epidemiological history, accelerated CKD, and unusual locations of urothelial carcinoma, the patient was diagnosed with BEN. Despite medical management and hemodialysis, the patient's renal function and mental status continued to deteriorate, and the decision was made to proceed with palliative care measures.

Isolation of single cells from individual mouse ovaries for flow cytometry and functional analysis.

Here, we present a validated workflow to isolate sufficient viable single ovary cells from a single mouse without the need to pool from several mice. We provide steps essential for estrous staging, ovary harvesting and dissociation, ovary cell staining, data collection, and analysis. Our approach allows the use of these single-cell suspensions for flow sorting, flow cytometry analysis, or functional in vitro assays. Importantly, our protocol is designed to maximize the isolation of immune cells, including T cell subsets.

IFN-γ and androgens disrupt mitochondrial function in murine myocytes.

The effect of cytokines on non-traditional immunological targets under conditions of chronic inflammation is an ongoing subject of study. Fatigue is a symptom often associated with autoimmune diseases. Chronic inflammatory response and activated cell-mediated immunity are associated with cardiovascular myopathies which can be driven by muscle weakness and fatigue. Thus, we hypothesize that immune dysfunction-driven changes in myocyte mitochondria may play a critical role in fatigue-related pathogenesis. We show that persistent low-level expression of IFN-γ in designated IFN-γ AU-Rich Element deletion mice (ARE mice) under androgen exposure resulted in mitochondrial and metabolic deficiencies in myocytes from male or castrated ARE mice. Most notably, echocardiography unveiled that low ejection fraction in the left ventricle post-stress correlated with mitochondrial deficiencies, explaining how heart function decreases under stress. We report that inefficiencies and structural changes in mitochondria, with changes to expression of mitochondrial genes, are linked to male-biased fatigue and acute cardiomyopathy under stress. Our work highlights how male androgen hormone backgrounds and active autoimmunity reduce mitochondrial function and the ability to cope with stress and how pharmacological blockade of stress signal protects heart function. These studies provide new insight into the diverse actions of IFN-γ in fatigue, energy metabolism, and autoimmunity. © 2023 The Pathological Society of Great Britain and Ireland. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.

Myeloid-Derived Suppressive Cell Expansion Promotes Melanoma Growth and Autoimmunity by Inhibiting CD40/IL27 Regulation in Macrophages.

The relationship between cancer and autoimmunity is complex. However, the incidence of solid tumors such as melanoma has increased significantly among patients with previous or newly diagnosed systemic autoimmune disease (AID). At the same time, immune checkpoint blockade (ICB) therapy of cancer induces de novo autoinflammation and exacerbates underlying AID, even without evident antitumor responses. Recently, systemic lupus erythematosus (SLE) activity was found to drive myeloid-derived suppressor cell (MDSC) formation in patients, a known barrier to healthy immune surveillance and successful cancer immunotherapy. Cross-talk between MDSCs and macrophages generally drives immune suppressive activity in the tumor microenvironment. However, it remains unclear how peripheral pregenerated MDSC under chronic inflammatory conditions modulates global macrophage immune functions and the impact it could have on existing tumors and underlying lupus nephritis. Here we show that pathogenic expansion of SLE-generated MDSCs by melanoma drives global macrophage polarization and simultaneously impacts the severity of lupus nephritis and tumor progression in SLE-prone mice. Molecular and functional data showed that MDSCs interact with autoimmune macrophages and inhibit cell surface expression of CD40 and the production of IL27. Moreover, low CD40/IL27 signaling in tumors correlated with high tumor-associated macrophage infiltration and ICB therapy resistance both in murine and human melanoma exhibiting active IFNγ signatures. These results suggest that preventing global macrophage reprogramming induced by MDSC-mediated inhibition of CD40/IL27 signaling provides a precision melanoma immunotherapy strategy, supporting an original and advantageous approach to treat solid tumors within established autoimmune landscapes. SIGNIFICANCE: Myeloid-derived suppressor cells induce macrophage reprogramming by suppressing CD40/IL27 signaling to drive melanoma progression, simultaneously affecting underlying autoimmune disease and facilitating resistance to immunotherapy within preexisting autoimmune landscapes.

The interplay of type I and type II interferons in murine autoimmune cholangitis as a basis for sex-biased autoimmunity.

We have reported on a murine model of autoimmune cholangitis, generated by altering the AU-rich element (ARE) by deletion of the interferon gamma (IFN-γ) 3' untranslated region (coined ARE-Del-/- ), that has striking similarities to human primary biliary cholangitis (PBC) with female predominance. Previously, we suggested that the sex bias of autoimmune cholangitis was secondary to intense and sustained type I and II IFN signaling. Based on this thesis, and to define the mechanisms that lead to portal inflammation, we specifically addressed the hypothesis that type I IFNs are the driver of this disease. To accomplish these goals, we crossed ARE-Del-/- mice with IFN type I receptor alpha chain (Ifnar1) knockout mice. We report herein that loss of type I IFN receptor signaling in the double construct of ARE-Del-/- Ifnar1-/- mice dramatically reduces liver pathology and abrogated sex bias. More importantly, female ARE-Del-/- mice have an increased number of germinal center (GC) B cells as well as abnormal follicular formation, sites which have been implicated in loss of tolerance. Deletion of type I IFN signaling in ARE-Del-/- Ifnar1-/- mice corrects these GC abnormalities, including abnormal follicular structure. CONCLUSION: Our data implicate type I IFN signaling as a necessary component of the sex bias of this murine model of autoimmune cholangitis. Importantly these data suggest that drugs that target the type I IFN signaling pathway would have potential benefit in the earlier stages of PBC. (Hepatology 2018;67:1408-1419).

Architecture of high-affinity unnatural-base DNA aptamers toward pharmaceutical applications.

We present a remodeling method for high-affinity unnatural-base DNA aptamers to augment their thermal stability and nuclease resistance, for use as drug candidates targeting specific proteins. Introducing a unique mini-hairpin DNA provides robust stability to unnatural-base DNA aptamers generated by SELEX using genetic alphabet expansion, without reducing their high affinity. By this method, >80% of the remodeled DNA aptamer targeting interferon-γ (KD of 33 pM) survived in human serum at 37 °C after 3 days under our experimental conditions, and sustainably inhibited the biological activity of interferon-γ.

IFN-gamma AU-rich element removal promotes chronic IFN-gamma expression and autoimmunity in mice.

We generated a mouse model with a 162 nt AU-rich element (ARE) region deletion in the 3' untranslated region (3'UTR) of the interferon-gamma (IFN-γ) gene that results in chronic circulating serum IFN-γ levels. Mice homozygous for the ARE deletion (ARE-Del) (-/-) present both serologic and cellular abnormalities typical of patients with systemic lupus erythematosus (SLE). ARE-Del(-/-) mice display increased numbers of pDCs in bone marrow and spleen. Addition of IFN-γ to Flt3-ligand (Flt3L) treated in vitro bone marrow cultures results in a 2-fold increase in pDCs with concurrent increases in IRF8 expression. Marginal zone B (MZB) cells and marginal zone macrophages (MZMs) are absent in ARE-Del(-/-) mice. ARE-Del(+/-) mice retain both MZB cells and MZMs and develop no or mild autoimmunity. However, low dose clodronate treatment in ARE-Del(+/-) mice specifically eliminates MZMs and promotes anti-DNA antibody development and glomerulonephritis. Our findings demonstrate the consequences of a chronic IFN-γ milieu on B220(+) cell types and in particular the impact of MZB cell loss on MZM function in autoimmunity. Furthermore, similarities between disease states in ARE-Del(-/-) mice and SLE patients suggest that IFN-γ may not only be a product of SLE but may be critical for disease onset and progression.

Murine beta-defensin 2 promotes TLR-4/MyD88-mediated and NF-kappaB-dependent atypical death of APCs via activation of TNFR2.

Mammalian antimicrobial peptides, including beta-defensins, represent an ancient arm of innate immunity designed to directly neutralize invading microbes. Previously, we demonstrated that murine beta-defensin 2 (mDF2beta) also acted as an endogenous ligand for TLR-4-activating maturation of dendritic cells (DCs). Herein, we report that this TLR-4 -dependent activation leads to induction of an atypical cell death that is unexpectedly exaggerated by the inhibition of caspases. Experiments using APCs with nonfunctional TNF-alpha or its receptors suggest that this is a NF-kappaB- and TNF-alpha-dependent process that does not require TNFR1. We demonstrate that mDF2beta triggers a TNFR2-mediated signaling cascade of "self-destruction" through up-regulation of membrane-bound TNF-alpha and TNFR2. This appears not to be an isolated phenomenon, as human synthetic beta-defenisn 3 was also able to activate and kill DCs. We propose that beta-defenins may play an important immunoregulatory role as controllers of the natural process of elimination of activated APCs.

Regulation of ITAM-positive receptors: role of IL-12 and IL-18.

Our previous studies have identified mechanisms by which cytokine production, blocked by Ly49G2 receptor cross-linking, can be overridden. In this study we analyzed the regulation of other ITAM-positive receptor signaling on NK, NKT, and T cells and characterized the biochemical pathways involved in this signaling. Our studies demonstrate that cross-linking of NKG2D and NK1.1 results in a synergistic NK IFN-gamma response when combined with IL-12 or IL-18. Examination of NKT- and T-cell responses demonstrated that cross-linking of NKG2D and CD3 resulted in potent synergy when combined with IL-12 and, to a lesser degree, with IL-18. We have now found that both the p38 MAP kinase and the ERK-dependent signal transduction pathways are required for the synergistic response. Further mechanistic examination of the synergy indicated a potent up-regulation of total IFN-gamma mRNA in the nuclear and the cytoplasmic compartment, but mRNA half-life was not affected. Fifteen minutes of IL-12 pretreatment was sufficient to result in maximal synergistic activation, indicating that the response of the cells to the IL-12 signal was rapid and immediate. Thus, our data demonstrate that multiple convergent signals maximize the innate immune response by triggering complementary biochemical signaling pathways.

TGF-beta and vitamin D3 utilize distinct pathways to suppress IL-12 production and modulate rapid differentiation of human monocytes into CD83+ dendritic cells.

We previously demonstrated that agents known to signal infection or inflammation can rapidly and directly drive differentiation of human CD14+ monocytes into CD83+ dendritic cells (DCs) when introduced to cells under serum-free conditions. In this study, we evaluated the effects of TGF-beta and vitamin D3 (VitD3) on the proportion and function of monocytes that adopt DC characteristics. TGF-beta significantly decreased the proportion of cells that rapidly adopted stable DC characteristics in response to LPS, but had little or no effect on calcium ionophore-induced differentiation. In contrast, VitD3 showed no such pathway specificity and dramatically suppressed differentiation of monocytes into DCs in response to these agents. Both TGF-beta and VitD3 altered cytokine and chemokine production in LPS-treated monocytes, inhibited IL-12 and IL-10 secretion, and decreased the functional capacity of DCs. Despite the similar effects of TGF-beta and VitD3, there are significant differences in the signaling pathways used by these agents, as evidenced by their distinct effects on LPS- and calcium ionophore-induced DC differentiation, on LPS-induced secretion of IL-10, and on two members of the NF-kappaB family of transcription factors, RelB and cRel. These studies identify TGF-beta and VitD3 as potent regulatory factors that use distinct pathways to suppress both the differentiation of DCs as well as their capacity to secrete the Th1-polarizing cytokine IL-12. Because these agents are present in serum and negatively affect DC differentiation at physiological concentrations, our findings are likely to have significance regarding the in vivo role of TGF-beta and VitD3 in determining the type of immune responses.