Autoimmunity and Cell Streaming

IFNγ is a Renovator of β cells

10/12/2019

The firm belief that Type 1 diabetes results from an inflammatory autoimmune attack has led researchers to explain their findings in ways that confirm this protected model. Any influence that supports inflammation and autoimmunity is expected to be problematic and any protective therapy is assumed to work against these factors. The helper T cell type 1 (Th1) response has been portrayed as the more aggressive and inflammatory branch of the adaptive immune system. As a result, it has been widely assumed responsible for the destruction of the β cells, which is believed to be mediated largely through its principal inflammatory cytokine, Interferon-gamma (IFNγ).

Most articles that invoke the β cell destructive capacity of IFNγ refer back to the early study by Sarvetnick et al. (1988), which showed that some BALB/c mice, in particular some males[1], with an IFNγ transgene linked to the insulin promoter (ins-IFNγ-tg/BALB/c) developed Type 1 diabetes[2]. Histological analysis of their pancreata showed variable infiltration of islets and surrounding tissue by inflammatory lymphocytes and macrophages. It was uncertain, however, why only less than half of the transgenic mice developed diabetes, but these findings were sufficient to brand IFNγ as a major driver of diabetes. Gu and Sarvetnick (1993) realized soon after that there was evidence for dramatic β cell regeneration from ductal cell precursors in the ins-IFNγ-tg/BALB/c strain, making these mice one of the earliest examples of adult β cell regeneration, which was previously considered impossible[3]. IFNγ was directing an autoimmune destruction of the β cells[4] while also stimulating their regeneration from the ductal cells. In the mice that did not progress to diabetes there was evidently a balance between these two functions, sufficient enough to maintain normoglycemia. Later, Sarvetnick’s group crossed the ins-IFNγ transgene to the diabetes-prone NOD background (ins-IFNγ-tg/NOD) and again saw an inflammatory autoimmune infiltration of the islets with continual destruction and regeneration of β cells, but these mice were protected from developing diabetes[5]. The propensity to develop diabetes in some ins-IFNγ-tg/BALB/c mice may then have been caused by some unique vulnerability in the male-BALB/c background that is not present in NOD mice, which are the closest animal model to human diabetes.

IFNγ’s multiple influences that potentiate the destruction of existing β cells has overshadowed its regenerative influence and its larger context as a mediator of tissue remodeling. IFNγ causes macrophages to increase their production of TNFα, IL-1, and IL-12, the latter of which further promotes the production of IFNγ by helper T cells and supports their differentiation to a Th1 profile[6]. IFNγ expression increases the production of addresins and adhesion molecules in the pancreas to recruit more lymphocytes[7]. The cytotoxicity, motility, speed, and abundance of CD8 T cells is enhanced by IFNγ exposure [8, 9]. IFNγ also has direct effects on β cells, increasing their expression of self antigens on MHC I [10], and impairing their differentiation[11]. While IFNγ does not seem to directly possess the capacity for inducing their apoptosis, it is capable of sensitizing the β cells to apoptotic signals[12] and to perforin mediated cell death[13], and has direct cytotoxic effects to β cells when combined with TNFα [14][15]. These factors considered, it is no wonder that IFNγ is considered a major driver of β cell loss. Especially when studied out of context in in vitro studies where β cells are isolated from the whole organ system, ductal cell regeneration is impossible and IFNγ only shows its destructive effect. The effect on streaming[16] of the ductal precursors into functioning β cells more fully completes the picture of what IFNγ can accomplish in the context of Type 1 diabetes.

The ins-IFNγ-tg/BALB/c mice showed a continual differentiation of ductal structures into islet structures, where ductal epithelial cells divided into cells co-expressing amylase and insulin, representing an intermediate ascinar/β cell phenotype[17]. Moving away from the lumen, these cells lost their amylase production, and migrated together to form new β cell containing islets. These observations mimic the process of islet development in ontogeny, and Zhang and Sarvetnick (2003) showed that the same regulatory molecules found in the developing pancreas were necessary to fulfill the regenerative stimulus of IFNγ[18]. In particular, Neurogenin 3 (NGN3) is a necessary and sufficient transcription factor for potentiating the endocrine differentiation of ductal cells. A complex of the pro-inflammatory cytokines IFNγ, TNFα, and IL-1β was seen to up-regulate NGN3 expression on ductal cells through STAT3 signaling[19]. It should also be noted that the ontological development of β cells occurs in a high IFNγ environment[20].

IFNγ’s dual action as both a destroyer and creator of β cells leads to variable results in studies manipulating its activity and complicates the perception of its role in diabetes. In the ins-IFNγ-tg/BALB/c male mice that do develop Type 1 diabetes, treatment with an anti-IFNγ antibody restores insulin production and normoglycemia[1]. Indeed, in these mice, since non-transgenic Balb/c do not regularly develop diabetes, it is certainly the transgenic expression of IFNγ that causes the diabetes in the subset of male mice. Since most transgenic mice and the females do not develop diabetes, the diabetogenic effect is likely representative of some unique problem in some male mice. A imbalance in favor of IFNγ’s pro-apoptotic effect compared to its regenerative effect, possibly influenced by the difference in sex hormones, would make IFNγ a net diabetogenic factor. The anti-IFNγ antibody is also successful at preventing cyclophosphamide and T cell transfer accelerated diabetes[21]. It is possible that cyclophosphamide or the process of T cell transfer are diabetogenic by pushing IFNγ towards the more destructive and less regenerative potential. In the development of spontaneous diabetes, genetic deletion of IFNγ production or treatment with anti-IFNγ antibody delay but do not prevent diabetes[22, 23]. Conversely, the injection of recombinant IFNγ completely prevents the development of spontaneous diabetes in NOD mice and in BB rats[24, 25]. IFNγ transgenic mice are resistant to streptozotocin-induced diabetes[26]. As already mentioned, NOD mice with the IFNγ transgene are resistant to spontaneous diabetes[5]. Both the CFA and BCG vaccines are protective through an IFNγ-dependent mechanism[27, 28]. The BCG vaccine has been used in a human diabetes trial with a temporary improvement of C-peptide[29], and another study showed a long-term increase of IFNγ after treatment[30].

The typical response to the unexpected evidence for a protective effect from IFNγ is to suggest that IFNγ is evidently exhibiting an anti-inflammatory and immunosuppressive capacity[25][15][24][31]. To support this, IFNγ’s ability to induce regulatory T cell activity or antagonize autoreactive effector functions as a counter regulatory measure is cited. However, Sarvetnick’s ins-IFNγ-tg/NOD mice are protected from diabetes while still showing a continual inflammatory autoimmune process[5]. In these mice, IFNγ expression is evidently participating in promoting the insulitis since IFNγ also causes insulitis in the ins-IFNγ-tg/BALB/c mice, which are not normally prone to diabetes[2]. On the other hand, mice and rats who are protected against diabetes by r-IFNγ show lower insulitis scores compared to non-treated controls, which has been argued to be the mechanism of protection[25][15][24][31]. But an anti-inflammatory effect of IFNγ may be an outcome instead of the mechanism of its protection. For example, a shift from a pro-inflammatory Th1 to anti-inflammatory Th2 cytokine profile after treatment with the diabetes-protective BCG and CFA vaccines is the outcome of an IFNγ-dependent protective mechanism rather than the cause of protection[27]. It should be expected that once the original threat is resolved and β cells are regenerated that the inflammatory response would discontinue.

Since IFNγ is produced by multiple kinds of cells, the protective effects of transgenic or injected IFNγ do not necessarily inform which kind of IFNγ producing immune response is desirable in Type 1 diabetes. IFNγ can be produced by natural killer (NK) cells and natural killer T (NKT) cells as part of the innate immune system or by Th1 and CD8 cells as part of the cell-mediated adaptive immunity. High levels of IFNγ are detected during the prediabetic autoimmune response, signifying a Th1-based response[32]. This period is also characterized by high β cell proliferation and a lack of symptoms[33]. Symptomatic onset of diabetes, on the other hand, is accompanied by a drop in IFNγ[34][35], suggesting that the Th1-mediated autoimmunity may have participated in preventing overt diabetes. The ins-IFNγ-tg/NOD mice show an autoimmune infiltration driven by IFNγ, implying a Th1 phenotype, and are protected from diabetes[5]. Further, GAD65-specific CD4 cells that secrete high amounts of IFNγ upon stimulation delay diabetes transfer when co-transfered along with splenocytes from diabetic NOD mice[36]. Various studies also indicate a defective production of IFNγ by Th1 cells in diabetics. Patients with newly diagnosed and longstanding Type 1 diabetes showed reduced levels of intracellular IFNγ in CD4 T-cells from phorbol-myristate acetate and ionomycin stimulated peripheral blood mononuclear cells (PBMCs)[37]. Another study reported a lower secretion of IFNγ from phytohemagglutinin-stimulated PBMCs at diagnosis of Type 1 diabetes in children and young adults [38]. Lohmann et al. (2002) also found a reduced secretion of IFNγ and TNFα from fresh PBMCs in children with newly diagnosed Type 1 diabetes [39]. However, mice with the IFNγ transgene on the glucagon promoter show that IFNγ is capable of inducing β cell remodeling, a non-diabetogenic balance of apoptosis and regeneration, without a lymphocytic infiltration[40]. This transgene was not backcrossed onto a diabetes-prone strain to test its protective potential, but it shows that IFNγ can fulfill the important remodeling effort without lymphocyte involvement, which suggests that the innate system alone may be able to satisfy this role. Defects are also seen in the IFNγ producing capacity of innate cells in diabetics. NK cells are reduced in number, are insensitive to activation, and LAK cells (activated NK cells) show reduced IFNγ secretion and cytotoxicity in diabetics [41]. Enhancement of NK cell cytotoxicity and IFNγ secretion through CFA treatment is protective against diabetes in NOD mice[42][43]. Similarly, NKT cells from NOD mice show a defect in proliferation and differentiation towards an IFNγ secreting phenotype upon T cell receptor engagement and IL-12 stimulation[44]. Altogether, the IFNγ capacity of the entire immune system appears to be important for diabetes protection and is defective in diabetics. Whether derived from the innate or adaptive system, the production of IFNγ represents the same general remodeling effort, with the antigen-specific Th1 response perhaps representing a more intensified and targeted response when cellular degeneration has exceeded the capacity of innate immune system.

Interferon gamma is a member of the interferon class of cytokines named after their ability to interfere with viral infection. Although still shrouded in uncertainty, perhaps the most popular suspect for triggering the “autoimmune attack” in Type 1 diabetes is a viral infection of the β cells, especially by the coxsackie B4 virus (CVB4). As a critical cytokine in the antiviral response, it is surprising that IFNγ has only scantily been considered as a possibly purposeful response to a diabetogenic virus[34]. IFNγ activates the Jak-STAT signaling pathway in β cells[45] and inhibits the generation of infectious CVB4, whereas in β cells that had IFN cytokine signaling suppressed, viral infection was not inhibited by IFNγ[46]. PBMCs isolated from children with Type 1 diabetes show an impaired capacity to produce IFNγ and other Th1 immune markers in response to CVB4[47]. During a viral infection, tissue remodeling is a protective mechanism where compromised cells are cleared and replaced by progenitor or stem cells [48], similar to what is seen in the ins-IFNγ-tg mice strains. Persistence of viral infection inhibits Th1 activity and leads to a decline and dysfunction of CD8 cells[49]. It is possible that viral persistence in the β cells after an unsuccessful immune response leads eventually to the impairment of of the Th1 immune response and overt diabetes onset. Although the evidence for a viral origin of diabetes is uncertain as of yet, the protective effects of IFNγ may be evidence supporting a viral etiology.

Whether or not a viral infection is involved, the benefit of a remodeling effort is suggestive that a global problem in the tissue needs to be overhauled by clearance and renovation. All tissues in the body are constantly streaming at different rates from precursor or stem cells to mature, differentiated cells[50]. In the event of some compromising stress, this renewal process can be hastened. In my article, “Autoimmunity is a Protective Response to β Cell Damage”, I proposed that the remodeling effort of IFNγ would need to resolve before the newly regenerated β cells could differentiate to their proper functioning. This, however, is not the case, as the constant IFNγ production and recycling of β cells can still allow for sufficient insulin production and glucose control. Either a defect in streaming of precursor cells or an unusually enhanced apoptotic sensitivity could push the renewal balance in favor of disease. The Th1 response is a dramatic effort that risks these possible complications in order to secure a renewed tissue. The evidence presented here suggests that the IFNγ, Th1-based response is critically protective, despite its aggressive nature.

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