Interferon-γ directly induces gastric epithelial cell death and is required for progression to metaplasia

Chronic inflammation of the gastric mucosa, often caused by autoimmune gastritis and/or infection with Helicobacter pylori, can lead to atrophy of acid-secreting parietal cells with metaplasia of remaining cells. The histological pattern marks a critical step in the progression from chronic gastritis to gastric cancer, yet underlying mechanism(s) of inflammation-induced cell death of gastric epithelial cells are poorly understood. We investigated direct effects of a type 1 cytokine associated with autoimmunity and infection, interferon-γ (IFN-γ), on gastric epithelial cells. IFN-γ was applied to three-dimensional organoid cultures of gastric epithelial cells derived from gastric corpus gland (gastroids) of control and IFN-γ receptor-deficient mice. Gastroids were also treated with supernatants from activated immune cells isolated from a mouse model of autoimmune-mediated atrophic gastritis (TxA23) with and without IFN-γ expression. Finally, histopathological analysis of atrophy and metaplasia severity was performed in TxA23 mice and compared to TxA23 × Ifng^−/− mice. Gastric epithelial cells in gastroid cultures expressed IFN-γ receptor in the basolateral membrane, and gastroids died when treated with IFN-γ in an IFN-γ receptor-dependent manner. Supernatants from immune cells containing high levels of IFN-γ were highly toxic to gastroids, and toxicity was tempered when IFN-γ was either neutralized using a monoclonal antibody or when supernatants from Ifng^−/− mouse immune cells were used. Finally, TxA23 × Ifng^−/− mice showed near-complete abrogation of pre-cancerous histopathological atrophy and metaplasia versus IFN-γ-sufficient controls. We identify IFN-γ as a critical promoter of parietal cell atrophy with metaplasia during the progression of gastritis to gastric atrophy and metaplasia. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

IFN-γ induction by neutrophil-derived IL-17A homodimer augments pulmonary antibacterial defense

The role of interleukin-17A (IL-17A) in host defense against Legionella pneumophila remains elusive. To address this issue, we used Il17a^−/−, Il17f^−/−, and Il17a/Il17f^−/− mice on a C57Bl/6 (non-permissive) background and IL-17 neutralizing Abs in mice on an A/J (permissive) background. Higher bacterial (L. pneumophila) counts in the lung and blood along with reduced neutrophil recruitment were detected in Il17a^−/−, but not Il17f^−/−, mice. We found that neutrophils produce IL-17A homodimer (IL-17A) during L. pneumophila infection, and hematopoietic cell-derived IL-17A is known to be important for bacterial clearance. Thus, intratracheal administration of wild-type neutrophils or recombinant IL-17A restored bacterial clearance and neutrophil recruitment in Il17a^−/− mice. Furthermore, neutrophil-depleted Rag2^−/− and Rag2/Il-2rγ^−/− mice exhibited increased bacterial burden, reduced neutrophil influx and IL-17A production in the lung. Recombinant IFN-γ administration in Il17a^−/− mice augmented bacterial elimination, whereas IL-17A administration in Ifnγ^−/− mice did not augment bacterial clearance. IFN-γ is produced by T cells, but not neutrophils or macrophages, suggesting that neutrophil-derived IL-17A induces IFN-γ in a paracrine fashion. Human pneumonic lungs and human neutrophils challenged with L. pneumophila exhibited increased numbers of IL-17A producing cells. These findings display a novel function of neutrophil-derived IL-17A in antibacterial defense via the induction of IFN-γ in a paracrine manner.     

Contrasting immune responses mediate Campylobacter jejuni-induced colitis and autoimmunity

Campylobacter jejuni is a leading cause of foodborne enteritis that has been linked to the autoimmune neuropathy, Guillain Barré syndrome (GBS). C57BL/6 interleukin (IL)-10^+/+ and congenic IL-10^−/− mice serve as C. jejuni colonization and colitis models, respectively, but a mouse model for GBS is lacking. We demonstrate that IL-10^−/− mice infected with a C. jejuni colitogenic human isolate had significantly upregulated type 1 and 17 but not type 2 cytokines in the colon coincident with infiltration of phagocytes, T cells and innate lymphoid cells (ILCs). Both ILC and T cells participated in interferon-γ (IFN-γ), IL-17, and IL-22 upregulation but in a time- and organ-specific manner. T cells were, however, necessary for colitis as mice depleted of Thy-1⁺ cells were protected while neither Rag1^−/− nor IL-10R blocked Rag1^−/− mice developed colitis after infection. Depleting IFN-γ, IL-17, or both significantly ameliorated colitis and drove colonic responses toward type 2 cytokine and antibody induction. In contrast, C. jejuni GBS patient strains induced mild colitis associated with blunted type 1/17 but enhanced type 2 responses. Moreover, the type 2 but not type 1/17 antibodies cross-reacted with peripheral nerve gangliosides demonstrating autoimmunity.     

Pam3CSK4 enhanced beta cell loss and diabetogenesis: The roles of IFN-gamma and IL-17

Toll like receptors are primary sensors of both innate and adaptive immune systems. They activate APCs and influence T-cell function in inflammatory autoimmune response. Studies have shown that TLR manipulation may lead to either tolerance or trigger autoimmunity. Using diabetogenic and subdiabetogenic multiple low doses of streptozotocin, we demonstrate here that Pam3 CYS-CK₄ a TLR-2 agonist, enhances and promotes diabetes in C57BL/6 male mice following increased apoptosis of β islet cells. FACS analysis of isolated pancreatic lymph node cells revealed significant increased number of macrophages, dendritic cells, CD4⁺ TNF-α⁺, CD4⁺ IFN-γ⁺ and most significantly, CD4⁺ IL-17⁺ and reduced number of CD25⁺Fox p3⁺ T cells after Pam3CSK₄ treatment. Genetic deletion of IFN-γ prevents whereas deletion of IL-17 reduced severity of Pam3CSK₄-induced enhancement of diabetes. TLR-2 agonist-enhanced diabetogenesis is also influenced by enhanced influx of antigen presenting cells and suppression of regulatory T cell activity.     

IFN-γ is reciprocally involved in the concurrent development of organ-specific autoimmunity in the liver and stomach

Interferon (IFN)-γ acts as a critical proinflammatory mediator in autoimmune processes, whereas it exerts regulatory functions to limit tissue damage associated with inflammation. However, a detailed understanding of the complex roles of IFN-γ in the development of organ-specific autoimmunity is still lacking. Recently, we found that programmed cell death 1-deficient mice thymectomized 3 days after birth (NTx–PD-1^{? / ?} mice) concurrently developed autoimmune hepatitis (AIH) and autoimmune gastritis (AIG). In this study, we investigated the roles of IFN-γ in the development of AIH and AIG in this mouse model. In NTx–PD-1^{? / ?} mice, serum levels of IFN-γ were markedly elevated. Neutralization of IFN-γ prevented the development of AIG. However, the same treatment exacerbated hepatic T-cell infiltration in AIH. Because of the loss of anti-proliferative effects by IFN-γ, neutralization of IFN-γ increased T-cell proliferation in the spleen and liver, resulting in exacerbated T-cell infiltration in the liver. On the other hand, in the development of AIG, CD4⁺ T-cell migration into the gastric mucosa is essential for induction. CCL20 expression was up-regulated in the gastric mucosa, and anti-CCL20 suppressed CD4⁺ T-cell infiltration into the gastric mucosa. Importantly, anti-IFN-γ suppressed CCL20 expression and infiltration of CD4⁺ T cells in the gastric mucosa, whereas in vivo injection of recombinant IFN-γ up-regulated CCL20 expression in the stomach, suggesting that IFN-γ is critically involved in CD4⁺ T-cell accumulation in AIG by up-regulating local CCL20 expression. In conclusion, IFN-γ is involved differently in the development of AIH and of AIG. IFN-γ negatively regulates T-cell proliferation in fatal AIH, whereas it initiates development of AIG. These findings imply that increased production of IFN-γ induced by an organ-specific autoimmunity may trigger the concurrent development of another organ-specific autoimmune disease.     

Disruption of the Notch pathway aggravates airway inflammation by inhibiting regulatory T cell differentiation via regulation of plasmacytoid dendritic cells

Plasmacytoid dendritic cells (pDCs) regulate immunity and promote tolerance in asthma. Notch signalling is a highly conserved pathway that regulates the immune response; however, its role in pDC-mediated asthmatic airway inflammation is unclear. This study clarified the effects of Notch signalling on pDC-mediated airway inflammation using murine models of ovalbumin-sensitized allergic asthma. RBP-J-deficient pDCs (RBP-J^−/− pDCs) were co-cultured with naïve CD4⁺ T cells and supernatants and T cell subtypes were analysed. RBP-J^−/− pDCs were intranasally transferred to the airways of ovalbumin-sensitized recipient mice. Lung samples of all mice were subjected to tests for histopathology, cytokine profile of bronchoalveolar lavage fluid, airway hyperactivity and expression of T helper type 1 (Th1)/Th2 cells, regulatory T cells and type 2 innate lymphoid cells (ILC2s). The results showed that pDCs with and without RBP-J deficiency significantly differed in expression levels of cluster of differentiation 83 (CD83), but not CD80, CD86 and major histocompatibility complex class II. Co-culturing pDCs with naïve T cells revealed a poorer immunosuppressive effect of RBP-J^−/− pDCs. This may be attributed to the lower expression levels of inducible co-stimulator ligand and lower production of interleukin 10 in RBP-J^−/− pDCs than in control pDCs, which impeded T cell activation and Treg suppression. RBP-J^−/− pDCs were associated with high ILC2 expression and severe Th2 immune responses and airway inflammation. Therefore, Notch signalling is critical for pDC-dependent immunoregulation, and RBP-J deficiency reduces pDC-based immunosuppression via T cell activation and Th cell differentiation. Thus, this pathway may be a therapeutic target for pDC-based anti-asthma immunotherapy.     

Successful treatment of murine autoimmune cholangitis by parabiosis: Implications for hematopoietic therapy

There is a significant unmet need in the treatment of primary biliary cirrhosis (PBC) despite significant data on the effector pathways that lead to biliary duct damage. We focused attention on a murine model of PBC, the dominant negative transforming growth factor β receptor II (Tg) mice. To further define the pathways that lead to biliary pathology in these mice, we developed Tg mice deleted of CD4 cells (CD4^−/−Tg).Interestingly, these mice developed more severe cholangitis than control Tg mice. These mice, which lack CD4 cells, manifested increased levels of IFN-γ produced by effector CD8 cells. It appears that increased cholangitis is due to the absence of CD4 Treg cells. Based on these data, we parabiosed CD4^−/−Tg mice with established disease at 8–9 weeks of age with C57BL/6 control mice. Such parabiotic “twins” had a significant reduction in autoimmune cholangitis, even though they had established pathology at the time of surgery. We prepared mixed bone marrow chimera mice constructed from CD4^−/−Tg and CD8^−/− mice and not only was cholangitis improved, but a decrease in terminally differentiated CD8⁺ T effector cells in the presence of wild type CD4 cells was noted. In conclusion, “correcting” the CD4 T cell subset, even in the presence of pathogenic CD8 T cells, is effective in treating autoimmune cholangitis.     

Interferon-γ is required during the initiation of an organ-specific autoimmune disease

Autoimmune gastritis induced by neonatal thymectomy of mice is a CD4⁺ T cell-mediated organ-specific autoimmune disease. The characteristic features of autoimmune gastritis, which include a mononuclear infiltrate within the gastric mucosa, loss of parietal and chief cells and circulating autoantibodies to the gastric H⁺/K⁺ ATPase, appear 6–10 weeks after thymectomy. Here we have assessed the role of interferon-γ (IFN-γ) in the pathogenesis of the gastric lesion. Splenic T cells derived from mice with gastritis produced three- to tenfold more IFN-γ than T cells from normal animals after stimulation with anti-CD3 antibodies. Treatment of neonatally thymectomized mice at weekly intervals for 6 or 12 weeks with a neutralizing rat monoclonal antibody to mouse IFN-γ abolished the production of anti-gastric autoantibodies and decreased the incidence of gastric mononuclear infiltrates from the 69% observed in normal rat immunoglobulin (Ig)-injected mice to 16%. Further, in mice treated with only a single dose of anti-IFN-γ immediately after thymectomy at 3 days after birth, the incidence of autoimmune gastritis was 1/19 compared to 8/19 in normal rat Ig-injected mice. Prevention of autoimmunity by neutralization of IFN-γ several weeks prior to the detection of a pathological lesion strongly suggests that IFN-γ plays an essential role in the initiation of the gastric autoimmune response.     

Sensory thresholds and the antinociceptive effects of GABA receptor agonists in mice lacking the β3 subunit of the GABAA receptor

A line of mice was recently created in which the gabrb3 gene, which encodes the β₃ subunit of the GABA_A receptor, was inactivated by gene-targeting. The existence of mice with a significantly reduced population of GABA_A receptors in the CNS enabled an investigation of the role of GABA and GABA_A receptors in nociception. The present study examined the sensory thresholds of these mice, as well as the antinociceptive effects of subcutaneously or intrathecally administered GABA_A and GABA_B receptor agonists. Homozygous null (β₃^−/−) mice displayed enhanced responsiveness to low-intensity thermal stimuli in the tail-flick and hot-plate test compared to C57BL/6J and 129/SvJ progenitor strain mice, and their wild-type (β₃^+/+) and heterozygous (β₃^+/−) littermates. The β₃^−/− mice also exhibited enhanced responsiveness to innocuous tactile stimuli compared to C57BL/6J, 129/SvJ and to their β₃^+/+ littermates as assessed by von Frey filaments. The presence of thermal hyperalgesia and tactile allodynia in β₃^−/− mice is consistent with a loss of inhibition mediated by presynaptic and postsynaptic GABA_A receptors in the spinal cord. As expected, subcutaneous administration of the GABA_A receptor agonist 4,5,6,7-tetrahydroisoxazolo-(5,4-c)pyridin-3-ol did not produce antinociception in β₃^−/− mice, whereas it produced a dose-dependent increase in hot-plate latency in C57BL/6J, 129/SvJ, β₃^+/+ and β₃^+/− mice. However, the antinociceptive effect of the GABA_B receptor agonist baclofen in the tail-flick and hot-plate tests was also reduced in β₃^−/− mice compared to the progenitor strains, β₃^+/+ or β₃^+/− mice after either subcutaneous or intrathecal administration. This finding was unexpected and suggests that a reduction in GABA_A receptors can affect the production of antinociception by other analgesic drugs as well.     

Pneumococcal DnaJ modulates dendritic cell-mediated Th1 and Th17 immune responses through Toll-like receptor 4 signaling pathway

Pneumococcal DnaJ was recently shown to be a potential protein vaccine antigen that induces strong Th1 and Th17 immune response against streptococcus pneumoniae infection in mice. However, how DnaJ mediates T cell immune response against S. pneumoniae infection has not been addressed. Here, we investigate whether DnaJ contributes to the development of T cell immunity through the activation of bone marrow-derived dendritic cells (BMDCs). We found that endotoxin-free recombinant DnaJ (rDnaJ) induced activation and maturation of BMDCs via recognition of Toll-like receptor 4 (TLR4) and activation of MAPKs, NF-κB and PI3K-Akt pathways. rDnaJ-treated BMDCs effectively stimulated naïve CD4⁺ T cells to secrete IFN-γ and IL-17A. Splenocytes from mice that were adoptively transferred with rDnaJ-pulsed BMDCs secreted higher levels of IFN-γ and IL-17A compared with those that received PBS-activated BMDCs. Splenocytes from TLR4^−/− mice immunized with rDnaJ produced lower levels of IFN-γ and IL-17A compared with those from wild type mice. Our findings indicate that DnaJ can induce Th1 and Th17 immune responses against S. pneumoniae through activation of BMDCs in a TLR4-dependent manner.     

FcRγ promotes T cell apoptosis in Fas-deficient mice

Deficiency of Fas or its ligand leads to lymphocyte accumulation, lymphadenopathy, splenomegaly, and autoimmunity in mice and humans. Although the Fas pathway is important for limiting the number of peripheral T cells, inactivation of other pro-apoptotic molecules can also perturb T cell homeostasis independently of and/or in concert with Fas deficiency. Here, we show that combined deficiency of Fas and the Fc receptor common γ signaling chain (FcRγ) results in worsened T cell accumulation in comparison with mice lacking Fas alone, with a particularly marked increase in the number of TCRαβ⁺CD4⁻CD8⁻ double negative (DN) T cells. LPR FcRγ^−/− mice exhibited reduced survival due to progressive lymphadenopathy. We further investigated the mechanisms whereby FcRγ deficiency promotes lymphoproliferative disease in Fas-mutant mice. Interestingly, there were no significant differences in T cell proliferation between LPR FcRγ^+/+ and LPR FcRγ^−/− mice in vivo and in vitro. However, FcRγ deletion resulted in significantly decreased peripheral T cell apoptosis. Importantly, the observed increase in apoptosis was restricted to a subset of FcRγ⁺ T cells. These T cells, but not those lacking FcRγ expression, exhibited increased activation of caspases 3 and 9, indicating a role for FcRγ in driving their apoptosis. FcRγ⁺ DN T cells also showed enhanced sensitivity to TCR restimulation-induced cell death (RICD) despite lacking Fas, suggesting that TCR stimulation of autoreactive T cells in vivo may serve to eliminate FcRγ⁺ T cells and thus exert partial control over lymphoproliferative disease. Hence, our data reveal a novel role for FcRγ in promoting peripheral T cell apoptosis in Fas-deficient mice, which may be of significant value in understanding autoimmune lymphoproliferative syndromes.     

Genetic deletion of soluble 5′-nucleotidase II reduces body weight gain and insulin resistance induced by a high-fat diet

We previously investigated whether inhibition of AMP-metabolizing enzymes could enhance AMP-activated protein kinase (AMPK) activation in skeletal muscle for the treatment of type 2 diabetes. Soluble 5′-nucleotidase II (NT5C2) hydrolyzes IMP and its inhibition could potentially lead to a rise in AMP to activate AMPK. In the present study, we investigated effects of NT5C2 deletion in mice fed a normal-chow diet (NCD) or a high-fat diet (HFD). On a NCD, NT5C2 deletion did not result in any striking metabolic phenotype. On a HFD however, NT5C2 knockout (NT5C2^−/−) mice displayed reduced body/fat weight gain, improved glucose tolerance, reduced plasma insulin, triglyceride and uric acid levels compared with wild-type (WT) mice. There was a tendency towards smaller and fewer adipocytes in epididymal fat from NT5C2^−/− mice compared to WT mice, consistent with a reduction in triglyceride content. Differences in fat mass under HFD could not be explained by changes in mRNA expression profiles of epididymal fat from WT versus NT5C2^−/− mice. However, rates of lipolysis tended to increase in epididymal fat pads from NT5C2^−/− versus WT mice, which might explain reduced fat mass. In incubated skeletal muscles, insulin-stimulated glucose uptake and associated signalling were enhanced in NT5C2^−/− versus WT mice on HFD, which might contribute towards improved glycemic control. In summary, NT5C2 deletion in mice protects against HFD-induced weight gain, adiposity, insulin resistance and associated hyperglycemia.     

PD-L1 and PD-L2 modulate airway inflammation and iNKT-cell-dependent airway hyperreactivity in opposing directions

Interactions of the inhibitory receptor programmed death-1 (PD-1) with its ligands, programmed death ligand (PD-L)1 and PD-L2, regulate T-cell activation and tolerance. In this study, we investigated the role of PD-L1 and PD-L2 in regulating invariant natural killer T (iNKT)-cell-mediated airway hyperreactivity (AHR) in a murine model of asthma. We found that the severity of AHR and airway inflammation is significantly greater in PD-L2^−/− mice compared with wild-type mice after either ovalbumin (OVA) sensitization and challenge or administration of α-galactosylceramide (α-GalCer). iNKT cells from PD-L2^−/− mice produced significantly more interleukin (IL)-4 than iNKT cells from control mice. Moreover, blockade of PD-L2 interactions of wild-type iNKT cells in vitro with monoclonal antibodies (mAbs) resulted in significantly enhanced levels of IL-4 production. In contrast, PD-L1^−/− mice showed significantly reduced AHR and enhanced production of interferon-γ (IFN-γ) by iNKT cells. iNKT-deficient Jα18^−/− mice reconstituted with iNKT cells from PD-L2^−/− mice developed high levels of AHR, whereas mice reconstituted with iNKT cells from PD-L1^−/− mice developed lower levels of AHR compared with control. As PD-L2 is not expressed on iNKT cells but rather is expressed on lung dendritic cells (DCs), in which its expression is upregulated by allergen challenge or IL-4, these findings suggest an important role of PD-L2 on lung DCs in modulating asthma pathogenesis. These studies also indicate that PD-L1 and PD-L2 have important but opposing roles in the regulation of AHR and iNKT-cell-mediated activation.     

IL-12hi Rapamycin-Conditioned Dendritic Cells Mediate IFN-γ–Dependent Apoptosis of Alloreactive CD4+ T Cells In Vitro and Reduce Lethal Graft-Versus-Host Disease

Rapamycin (RAPA) inhibits the mechanistic target of rapamycin (mTOR), a crucial immune system regulator. Dendritic cells (DC) generated in RAPA (RAPA-DC) enrich for CD4⁺ forkhead box p3 (FoxP3⁺) regulatory T cells and induce T cell apoptosis by an unknown mechanism. RAPA-DC also promote experimental allograft survival, yet paradoxically secrete increased IL-12, crucial for the generation of IFN-γ⁺ CD4⁺ T cells. However, IFN-γ is pro-apoptotic and IL-12-driven IFN-γ inhibits experimental graft-versus-host disease (GVHD). We hypothesized that IL-12^hi RAPA-DC would facilitate IFN-γ-mediated apoptosis of alloreactive T cells and, unlike control (CTR)-DC, would reduce lethal GVHD. Following LPS stimulation, RAPA-DC exhibited decreased MHCII and co-stimulatory molecules and contained a significant population of CD86^lo IL-12^hi cells. Consistent with our hypothesis, both unstimulated and LPS-stimulated RAPA-DC enhanced alloreactive CD4⁺ T cell apoptosis in culture. Augmented T cell apoptosis was ablated by IFN-γ neutralization or using T cells lacking the IFN-γ receptor, and it was associated with increased expression of Fas and cleaved caspase 8. DC production or responses to IFN-γ were not important to increased apoptotic functions of RAPA-DC. LPS-stimulated IL-12p40^−/− RAPA-DC induced lower levels of T cell apoptosis in culture, which was further decreased with addition of anti-IFN-γ. Finally, whereas CTR-DC accelerated mortality from GVHD, LPS-treated RAPA-DC significantly prolonged host survival. In conclusion, increased apoptosis of allogeneic CD4⁺ T cells induced by LPS-stimulated IL-12^hi RAPA-DC is mediated in vitro through IFN-γ and in part by increased IL-12 expression. Enhanced production of IL-12, the predominant inducer of IFN-γ by immune cells, is a probable mechanism underlying the capacity of LPS-treated RAPA-DC to reduce GVHD.     

The medulla controls effector primed γδT-cell development in the adult mouse thymus

γδT cells are produced in the thymus throughout life and provide immunity at epithelial-rich sites. Unlike conventional αβT cells, γδT-cell development involves intrathymic acquisition of effector function, with priming for either IL17 or IFN-γ production occurring during embryonic or adult life, respectively. How the thymus controls effector-primed γδT-cell generation in adulthood is poorly understood. Here, we distinguished de novo γδT cells from those undergoing thymus recirculation and/or retention using Rag2GFP mice alongside markers of maturation/effector priming including CD24, CD25, CD73, and IFN-γ, the latter by crossing with IFN-γ^YFP GREAT mice. We categorize newly developing γδT-cells into an ordered sequence where CD25⁺CD73⁻IFN-γ^YFP− precursors are followed sequentially by CD25⁻CD73⁺IFN-γ^YFP− intermediates and CD25⁻CD73⁺IFN-γ^YFP+ effectors. To determine intrathymic requirements controlling this sequence, we examined γδT-cell development in Relb^−/− thymus grafts that lack medullary microenvironments. Interestingly, medulla deficiency did not alter CD25⁺ γδT-cell precursor generation, but significantly impaired development of effector primed stages. This impact on γδT-cell priming was mirrored in plt/plt mice lacking the medullary chemoattractants CCL19 and CCL21, and also Ccl21a^−/− but not Ccl19^−/− mice. Collectively, we identify the medulla as an important site for effector priming during adult γδT-cell development and demonstrate a specific role for the medullary epithelial product CCL21 in this process.