An IFNγ-dependent immune-endocrine circuit lowers blood glucose to potentiate the innate antiviral immune response.

Viral infection makes us feel sick as the immune system alters systemic metabolism to better fight the pathogen. The extent of these changes is relative to the severity of disease. Whether blood glucose is subject to infection-induced modulation is mostly unknown. Here we show that strong, nonlethal infection restricts systemic glucose availability, which promotes the antiviral type I interferon (IFN-I) response. Following viral infection, we find that IFNγ produced by γδ T cells stimulates pancreatic ß cells to increase glucose-induced insulin release. Subsequently, hyperinsulinemia lessens hepatic glucose output. Glucose restriction enhances IFN-I production by curtailing lactate-mediated inhibition of IRF3 and NF-κB signaling. Induced hyperglycemia constrained IFN-I production and increased mortality upon infection. Our findings identify glucose restriction as a physiological mechanism to bring the body into a heightened state of responsiveness to viral pathogens. This immune-endocrine circuit is disrupted in hyperglycemia, possibly explaining why patients with diabetes are more susceptible to viral infection.

Difficulties in Accessing the List and Full Text of the Defended PhD Theses from Medical Schools: a Retrospective Case Study from Croatia.

OBJECTIVE: To conduct scientometric studies on PhD ("Doctor of Philosophy") theses (i.e., doctoral theses), researchers should be able to access the theses. We aimed to explore how to obtain a list and full text of the defended PhD theses from medical schools in Croatia over 30 years (from the beginning of 1992 to the end of 2021). METHODS: We tried to obtain information from the Croatian Bureau of Statistics, the National and University Library in Zagreb (NSK), universities, medical schools and online repositories. RESULTS: We could not find a single list (source) of all PhD theses. Based on 4 different sources (website of the University of Zagreb and Medical School in Rijeka; school administrator from Split; library catalog from Osijek), we gathered information that from the beginning of 1992 to the end of 2021, there were 2955 PhD theses defended at medical schools in Croatia - 357 in Osijek, 550 in Rijeka, 337 in Split and 1711 in Zagreb. In May 2022, the online Croatian Digital Dissertations Repository contained 631 (22%) of full-text theses in Portable Document Format (PDF). University of Zagreb School of Medicine has its own repository that holds the full text of 834 (49%) of their PhD theses. One of the three PhD programs of the University of Split School of Medicine, namely Translational Research in Biomedicine (TRIBE), published full texts of all PhD theses defended at that program on its website. NSK held 2650 (90%) of the theses in a printed version. CONCLUSION: It was extremely challenging to access the list and full texts of doctoral theses defended in Croatia. Making PhD theses publicly available would ensure transparency and enable analyses that should improve scientific policy.

The "Domino effect" in MASLD: The inflammatory cascade of steatohepatitis.

Metabolic dysfunction-associated steatotic liver disease (MASLD) is an increasingly common complication of obesity, affecting over a quarter of the global adult population. A key event in the pathophysiology of MASLD is the development of metabolic-associated steatohepatitis (MASH), which greatly increases the chances of developing cirrhosis and hepatocellular carcinoma. The underlying cause of MASH is multifactorial, but accumulating evidence indicates that the inflammatory process in the hepatic microenvironment typically follows a pattern that can be roughly divided into three stages: (1) Detection of hepatocyte stress by tissue-resident immune cells including γδ T cells and CD4-CD8- double-negative T cells, followed by their secretion of pro-inflammatory mediators, most notably IL-17A. (2) Recruitment of pro-inflammatory cells, mostly of the myeloid lineage, and initiation of inflammation through secretion of effector-type cytokines such as TNF, TGF-ß, and IL-1ß. (3) Escalation of the inflammatory response by recruitment of lymphocytes including Th17, CD8 T, and B cells leading to chronic inflammation, hepatic stellate cell activation, and fibrosis. Here we will discuss these three stages and how they are consecutively linked like falling domino tiles to the pathophysiology of MASH. Moreover, we will highlight the clinical potential of inflammation as a biomarker and therapeutic target for the treatment of MASLD.

NKG2D-mediated detection of metabolically stressed hepatocytes by innate-like T cells is essential for initiation of NASH and fibrosis.

Metabolic-associated fatty liver disease (MAFLD) is a spectrum of clinical manifestations ranging from benign steatosis to cirrhosis. A key event in the pathophysiology of MAFLD is the development of nonalcoholic steatohepatitis (NASH), which can potentially lead to fibrosis and hepatocellular carcinoma, but the triggers of MAFLD-associated inflammation are not well understood. We have observed that lipid accumulation in hepatocytes induces expression of ligands specific to the activating immune receptor NKG2D. Tissue-resident innate-like T cells, most notably γδ T cells, are activated through NKG2D and secrete IL-17A. IL-17A licenses hepatocytes to produce chemokines that recruit proinflammatory cells into the liver, which causes NASH and fibrosis. NKG2D-deficient mice did not develop fibrosis in dietary models of NASH and had a decreased incidence of hepatic tumors. The frequency of IL-17A+ γδ T cells in the blood of patients with MAFLD correlated directly with liver pathology. Our findings identify a key molecular mechanism through which stressed hepatocytes trigger inflammation in the context of MAFLD.

Largely preserved functionality after the combined loss of NKG2D, NCR1 and CD16 demonstrates the remarkable plasticity of NK cell responsiveness.

Natural killer (NK) cells play an important role in the early defense against tumors and virally infected cells. Their function is thought to be controlled by the balance between activating and inhibitory receptors, which often compete for the same ligands. Several activating receptors expressed on virtually all NK cells lack an inhibitory partner, most notably CD16, NCR1 and NKG2D. We therefore hypothesized that a signal through at least one of these receptors is always required for full NK cell activation. We generated animals lacking all three receptors (TKO) and analyzed their NK cells. In vitro, TKO NK cells did not show reduced ability to kill tumor targets but displayed hyperresponsiveness to NK1.1 stimulation. In vivo, TKO animals had a minor reduction in their ability to control non-hematopoietic tumors and cytomegalovirus infection, which was the result of reduced NK cell activity. Together, our findings show that activating NK cell receptors without an inhibitory partner do not provide a 'master' signal but are integrated in the cumulative balance of activating and inhibitory signals. Their activity is controlled through regulation of the responsiveness and expression of other activating receptors. Our findings may be important for future development of NK cell-based cancer immunotherapy.

Activation of Granulocytes in Response to a High Protein Diet Leads to the Formation of Necrotic Lesions in the Liver.

In their aspiration to become healthy, people are known to follow extreme diets. However, the acute impact on organs regulating systemic metabolism is not well characterized. Here, we investigated the acute impact of six extreme diets on the liver in mice. Most diets did not lead to clear pathology after short-term feeding. However, two weeks of feeding with a high protein diet (HPD) resulted in an acute increase of liver enzymes in the blood, indicative of liver damage. Histology revealed the formation of necrotic lesions in this organ which persisted for several weeks. Flow cytometric analysis of hepatic immune cell populations showed that HPD feeding induced activation of macrophages and neutrophils. Neutralization of the pro-inflammatory cytokine IL-1ß or depletion of macrophages with clodronate-loaded liposomes or with genetic models did not ameliorate liver necrosis. In contrast, the depletion of neutrophils prevented HPD-induced hepatic inflammation. After prolonged feeding, HPD-feeding was associated with a strong increase of the cytokines IL-10 and IL-27, suggesting that anti-inflammatory mediators are activated to prevent nutrient-overload-induced damage to the liver. In summary, whereas our data indicates that most extreme diets do not have a major impact on the liver within two weeks, diets with a very high protein content may lead to severe, acute hepatic damage and should therefore be avoided.

Extreme anaerobic exercise causes reduced cytotoxicity and increased cytokine production by peripheral blood lymphocytes.

Exercise has many beneficial effects for our body, but can become detrimental at high intensity, especially for our immune system. Little is known about the underlying mechanism of impaired immune functionality under conditions of intense physical strain. Freedivers, people who dive to high depths on a single breath, perform extreme exercise under anaerobic conditions. In this study, we investigated the impact of freediving on the cytotoxic arm of the immune system. At rest, elite freedivers did not display changes in their immunological profile compared to non-diving controls. In contrast, after a freedive, granzyme B and IL-2 production were reduced, whereas IFNγ and TNF secretion were increased by cytotoxic immune cells. Using in vitro models mimicking freedive conditions, we could show that hypoxia in combination with stress hyperglycemia had a negative impact on Granzyme B secretion, whereas IL-2 production was inhibited by stress hormones. Our findings suggest that in response to extreme exercise, cytotoxic immune cells transiently change their functional profile to limit tissue damage.

Hyperglycemia and Not Hyperinsulinemia Mediates Diabetes-Induced Memory CD8 T-Cell Dysfunction.

Type 2 diabetes (T2D) causes an increased risk of morbidity and mortality in response to viral infection. T2D is characterized by hyperglycemia and is typically associated with insulin resistance and compensatory hyperinsulinemia. CD8 T cells express the insulin receptor, and previously, we have shown that insulin is able to directly modulate effector CD8 T-cell function. We therefore hypothesized that memory CD8 T-cell responsiveness in the context of T2D is negatively impacted by hyperinsulinemia or hyperglycemia. Using a mouse model for T2D, we could show that memory CD8 T-cell function was significantly reduced in response to rechallenge by viral infection or with melanoma cells. Basal insulin injection of mice increased GLUT-1 expression and glucose uptake in memory CD8 T-cell precursors early after infection, which was prevented when these cells were deficient for the insulin receptor. However, neither insulin injection nor insulin receptor deficiency resulted in a difference in metabolism, memory formation, cytokine production, or recall responses of memory CD8 T cells compared with controls. Importantly, in context of obesity, insulin receptor deficiency on CD8 T cells did not affect the functional capacity of memory CD8 T cells. In contrast, we could show in vitro and in vivo that hyperglycemia significantly impairs the antiviral capacity of memory CD8 T cells. Our findings indicate that obesity impairs the memory CD8 T-cell response against viral infection and cancer through the detrimental effects of hyperglycemia rather than hyperinsulinemia.

Blood glucose regulation in context of infection.

The immune and endocrine systems collectively control homeostasis in the body. The endocrine system ensures that values of essential factors and nutrients such as glucose, electrolytes and vitamins are maintained within threshold values. The immune system resolves local disruptions in tissue homeostasis, caused by pathogens or malfunctioning cells. The immediate goals of these two systems do not always align. The immune system benefits from optimal access to nutrients for itself and restriction of nutrient availability to all other organs to limit pathogen replication. The endocrine system aims to ensure optimal nutrient access for all organs, limited only by the nutrients stores that the body has available. The actual state of homeostatic parameters such as blood glucose levels represents a careful balance based on regulatory signals from the immune and endocrine systems. This state is not static but continuously adjusted in response to changes in the current metabolic needs of the body, the amount of resources it has available and the level of threats it encounters. This balance is maintained by the ability of the immune and endocrine systems to interact and co-regulate systemic metabolism. In context of metabolic disease, this system is disrupted, which impairs functionality of both systems. The failure of the endocrine system to retain levels of nutrients such as glucose within threshold values impairs functionality of the immune system. In addition, metabolic stress of organs in context of obesity is perceived by the immune system as a disruption in local homeostasis, which it tries to resolve by the excretion of factors which further disrupt normal metabolic control. In this chapter, we will discuss how the immune and endocrine systems interact under homeostatic conditions and during infection with a focus on blood glucose regulation. In addition, we will discuss how this system fails in the context of metabolic disease.

NK cell receptor NKG2D enforces proinflammatory features and pathogenicity of Th1 and Th17 cells.

NKG2D is a danger sensor expressed on different subsets of innate and adaptive lymphocytes. Despite its established role as a potent activator of the immune system, NKG2D-driven regulation of CD4+ T helper (Th) cell-mediated immunity remains unclear. In this study, we demonstrate that NKG2D modulates Th1 and proinflammatory T-bet+ Th17 cell effector functions in vitro and in vivo. In particular, NKG2D promotes higher production of proinflammatory cytokines by Th1 and T-bet+ Th17 cells and reinforces their transcription of type 1 signature genes, including Tbx21. Conditional deletion of NKG2D in T cells impairs the ability of antigen-specific CD4+ T cells to promote inflammation in vivo during antigen-induced arthritis and experimental autoimmune encephalomyelitis, indicating that NKG2D is an important target for the amelioration of Th1- and Th17-mediated chronic inflammatory diseases.

Eomes broadens the scope of CD8 T-cell memory by inhibiting apoptosis in cells of low affinity.

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.

Loss of NKG2D in murine NK cells leads to increased perforin production upon long-term stimulation with IL-2.

NK cells are innate lymphocytes responsible for lysis of pathogen-infected and transformed cells. One of the major activating receptors required for target cell recognition is the NK group 2D (NKG2D) receptor. Numerous reports show the necessity of NKG2D for effective tumor immune surveillance. Further studies identified NKG2D as a key element allowing tumor immune escape. We here use a mouse model with restricted deletion of NKG2D in mature NKp46+ cells (NKG2DΔNK ). NKG2DΔNK NK cells develop normally, have an unaltered IFN-γ production but kill tumor cell lines expressing NKG2D ligands (NKG2DLs) less efficiently. However, upon long-term stimulation with IL-2, NKG2D-deficient NK cells show increased levels of the lytic molecule perforin. Thus, our findings demonstrate a dual function of NKG2D for NK cell cytotoxicity; while NKG2D is a crucial trigger for cytotoxicity of tumor cells expressing activating ligands it is also capable to limit perforin production in IL-2 activated NK cells.

'Beauty and the beast' in infection: How immune-endocrine interactions regulate systemic metabolism in the context of infection.

The immune and endocrine systems ensure two vital functions in the body. The immune system protects us from lethal pathogens, whereas the endocrine system ensures proper metabolic function of peripheral organs by regulating systemic homeostasis. These two systems were long thought to operate independently. The immune system uses cytokines and immune receptors, whereas the endocrine system uses hormones to regulate metabolism. However, recent findings show that the immune and endocrine systems closely interact, especially regarding regulation of glucose metabolism. In response to pathogen encounter, cytokines modify responsiveness of peripheral organs to endocrine signals, resulting in altered levels of blood hormones such as insulin, which promotes the ability of the body to fight infection. Here we provide an overview of recent literature describing various mechanisms, which the immune system utilizes to modify endocrine regulation of systemic metabolism. Moreover, we will describe how these immune-endocrine interactions derail in the context of obesity. From a clinical perspective we will elaborate how infection and obesity aggravate the development of metabolic diseases such as diabetes mellitus type 2 in humans. In summary, this review provides a comprehensive overview of immune-induced changes in systemic metabolism following infection, with a focus on regulation of glucose metabolism.

Cheating the Hunger Games; Mechanisms Controlling Clonal Diversity of CD8 Effector and Memory Populations.

Effector and memory CD8 T cells have an intrinsic difference in the way they must approach antigen; effector cells need to address the pathogen at hand and therefore favor outgrowth of only high-affinity clones. In contrast, the memory pool benefits from greater clonal diversity to recognize and eliminate pathogens with mutations in their immunogenic epitopes. Effector and memory fates are ultimately the result of the same three signals that control T cell activation; T cell receptor (TCR) engagement together with co-stimulation and cytokines. Great progress has been made in our understanding of the transcriptional programs that drive effector or memory differentiation. However, how these two different programs result from the same initial cues is still a matter of debate. An emerging image is that not only the classical three signals determine T cell differentiation, but also the ability of cells to access these signals relative to that of other activated clones. Inter-clonal competition is therefore not only a selective force, but also a mediator of CD8 T cell fate. How this is regulated on a transcriptional level, especially in the context of a selective "hunger game" based on antigen-affinity in which only cells of high-affinity are supposed to survive, is still poorly defined. In this review, we discuss recent literature that illustrates how antigen-affinity dependent inter-clonal competition shapes effector and memory populations in an environment of antigen affinity-driven selection. We argue that fine-tuning of TCR signal intensity presents an attractive target for regulating the scope of CD8 T cell vaccines.

NK cell receptor NKG2D sets activation threshold for the NCR1 receptor early in NK cell development.

The activation of natural killer (NK) cells depends on a change in the balance of signals from inhibitory and activating receptors. The activation threshold values of NK cells are thought to be set by engagement of inhibitory receptors during development. Here, we found that the activating receptor NKG2D specifically set the activation threshold for the activating receptor NCR1 through a process that required the adaptor DAP12. As a result, NKGD2-deficient (Klrk1-/-) mice controlled tumors and cytomegalovirus infection better than wild-type controls through the NCR1-induced production of the cytokine IFN-γ. Expression of NKG2D before the immature NK cell stage increased expression of the adaptor CD3ζ. Reduced expression of CD3ζ in Klrk1-/- mice was associated with enhanced signal transduction through NCR1, and CD3ζ deficiency resulted in hyper-responsiveness to stimulation via NCR1. Thus, an activating receptor developmentally set the activity of another activating receptor on NK cells and determined NK cell reactivity to cellular threats.

Virus-Induced Interferon-γ Causes Insulin Resistance in Skeletal Muscle and Derails Glycemic Control in Obesity.

Pro-inflammatory cytokines of a T helper-1-signature are known to promote insulin resistance (IR) in obesity, but the physiological role of this mechanism is unclear. It is also unknown whether and how viral infection induces loss of glycemic control in subjects at risk for developing diabetes mellitus type 2 (DM2). We have found in mice and humans that viral infection caused short-term systemic IR. Virally-induced interferon-γ (IFN-γ) directly targeted skeletal muscle to downregulate the insulin receptor but did not cause loss of glycemic control because of a compensatory increase of insulin production. Hyperinsulinemia enhanced antiviral immunity through direct stimulation of CD8+ effector T cell function. In pre-diabetic mice with hepatic IR caused by diet-induced obesity, infection resulted in loss of glycemic control. Thus, upon pathogen encounter, the immune system transiently reduces insulin sensitivity of skeletal muscle to induce hyperinsulinemia and promote antiviral immunity, which derails to glucose intolerance in pre-diabetic obese subjects. VIDEO ABSTRACT.

NKG2D: A Master Regulator of Immune Cell Responsiveness.

NKG2D is an activating receptor that is mostly expressed on cells of the cytotoxic arm of the immune system. Ligands of NKG2D are normally of low abundance, but can be induced in virtually any cell in response to stressors, such as infection and oncogenic transformation. Engagement of NKG2D stimulates the production of cytokines and cytotoxic molecules and traditionally this receptor is, therefore, viewed as a molecule that mediates direct responses against cellular threats. However, accumulating evidence indicates that this classical view is too narrow. During NK cell development, engagement of NKG2D has a long-term impact on the expression of NK cell receptors and their responsiveness to extracellular cues, suggesting a role in NK cell education. Upon chronic NKG2D engagement, both NK and T cells show reduced responsiveness of a number of activating receptors, demonstrating a role of NKG2D in induction of peripheral tolerance. The image that emerges is that NKG2D can mediate both inhibitory and activating signals, which depends on the intensity and duration of ligand engagement. In this review, we provide an overview of the impact of NKG2D stimulation during hematopoietic development and during acute and chronic stimulation in the periphery on responsiveness of other receptors than NKG2D. We propose that NKG2D interprets the context of the immunological environment through detection of cellular cues and in response sets the appropriate activation threshold for a large number of immune receptors. This perspective is of particular importance for future therapies that aim to exploit NKG2D signaling to fight tumors or infection.

NKG2D-Dependent Antitumor Effects of Chemotherapy and Radiotherapy against Glioblastoma.

Purpose: NKG2D is a potent activating immune cell receptor, and glioma cells express the cognate ligands (NKG2DL). These ligands are inducible by cellular stress and temozolomide (TMZ) or irradiation (IR), the standard treatment of glioblastoma, could affect their expression. However, a role of NKG2DL for the efficacy of TMZ and IR has never been addressed.Experimental Design: We assessed the effect of TMZ and IR on NKG2DL in vitro and in vivo in a variety of murine and human glioblastoma models, including glioma-initiating cells, and a cohort of paired glioblastoma samples from patients before and after therapy. Functional effects were studied with immune cell assays. The relevance of the NKG2D system for the efficacy of TMZ and IR was assessed in vivo in syngeneic orthotopic glioblastoma models with blocking antibodies and NKG2D knockout mice.Results: TMZ or IR induced NKG2DL in vitro and in vivo in all glioblastoma models, and glioblastoma patient samples had increased levels of NKG2DL after therapy with TMZ and IR. This enhanced the immunogenicity of glioma cells in a NGK2D-dependent manner, was independent from cytotoxic or growth inhibitory effects, attenuated by O6-methylguanine-DNA-methyltransferase (MGMT), and required the DNA damage response. The survival benefit afforded by TMZ or IR relied on an intact NKG2D system and was decreased upon inhibition of the NKG2D pathway.Conclusions: The immune system may influence the activity of convential cancer treatments with particular importance of the NKG2D pathway in glioblastoma. Our data provide a rationale to combine NKG2D-based immunotherapies with TMZ and IR. Clin Cancer Res; 24(4); 882-95. ©2017 AACR.

Efficient Killing of Murine Pluripotent Stem Cells by Natural Killer (NK) Cells Requires Activation by Cytokines and Partly Depends on the Activating NK Receptor NKG2D.

Natural killer (NK) cells play an important role as cytotoxic effector cells, which scan the organism for infected or tumorigenic cells. Conflicting data have been published whether NK cells can also kill allogeneic or even autologous pluripotent stem cells (PSCs) and which receptors are involved. A clarification of this question is relevant since an activity of NK cells against PSCs could reduce the risk of teratoma growth after transplantation of PSC-derived grafts. Therefore, the hypothesis has been tested that the activity of NK cells against PSCs depends on cytokine activation and specifically on the activating NK receptor NKG2D. It is shown that a subcutaneous injection of autologous iPSCs failed to activate NK cells against these iPSCs and can give rise to teratomas. In agreement with this result, several PSC lines, including two iPSC, two embryonic stem cell (ESC), and two so-called multipotent adult germline stem cell (maGSC) lines, were largely resistant against resting NK cells although differences in killing were found at low level. All PSC lines were killed by interleukin (IL)-2-activated NK cells, and maGSCs were better killed than the other PSC types. The PSCs expressed ligands of the activating NK receptor NKG2D and NKG2D-deficient NK cells from Klrk1-/- mice were impaired in their cytotoxic activity against PSCs. The low-cytotoxic activity of resting NK cells was almost completely dependent on NKG2D. The cytotoxic activity of IL-2-activated NKG2D-deficient NK cells against PSCs was reduced, indicating that also other activating receptors on cytokine-activated NK cells must be engaged by ligands on PSCs. Thus, NKG2D is an important activating receptor involved in killing of murine PSCs. However, NK cells need to be activated by cytokines before they efficiently target PSCs and then also other NK receptors become relevant. These features of NK cells might be relevant for transplantation of PSC-derived grafts since NK cells have the capability to kill undifferentiated cells, which might be present in grafts in trace amounts.