OPINION STATEMENT:Diabetes mellitus (DM) and its associated complications are becoming increasingly prevalent. Gastrointestinal symptoms associated with diabetes is known as diabetic enteropathy (DE) and may manifest as either diarrhea, fecal incontinence, constipation, dyspepsia, nausea, and vomiting or a combination of symptoms. The long-held belief that vagal autonomic neuropathy is the primary cause of DE has recently been challenged by newer theories of disease development. Specifically, hyperglycemia and the resulting oxidative stress on neural networks, including the nitrergic neurons and interstitial cells of Cajal (ICC), are now believed to play a central role in the development of DE. DE occurs in the majority of patients with diabetes; however, tools for early diagnosis and targeted therapy to counter the detrimental and potentially irreversible effects on the small bowel are lacking. Delay in diagnosis is further compounded by the fact that DE symptoms overlap with those of gastroparesis or can be confused with side effects from diabetes medications. Still, early recognition of the presence of DE is essential to mitigating symptoms and preventing further progression of complications including dysmotility and malabsorption. Current diagnostic modalities include manometry, wireless motility capsule (SmartPill™), and scintigraphy; however, these are not regularly utilized in clinical practice due to limited availability. Several medications are available for symptom relief in DE patients including rifaximin for small intestinal bacterial overgrowth (SIBO) and somatostatin analogues for diarrhea. While rodent models on stem cell therapy and alteration of the microbiome are promising, there is still a great need for further research on the pathologic underpinnings and development of novel treatment modalities for DE.

ObjectiveIncreased levels of type I interferon (IFN) and type I IFN–regulated genes are found in patients with systemic lupus erythematosus (SLE) and may be central to its pathogenesis. Mitochondrial antiviral signaling protein (MAVS) is a key regulator of type I IFN that undergoes a dramatic prion‐like aggregation and self propagates the activation signal from viral RNA to amplify downstream IFN production. We undertook this study to determine whether such MAVS aggregates might play a role in the sustained increased production of type I IFN in SLE.MethodsPeripheral blood mononuclear cells were isolated and mitochondrial extracts were prepared. MAVS aggregation was detected by semidenatured agarose gel electrophoresis and confirmed by immunofluorescence staining. MAVS‐associated signaling proteins were analyzed by Western blotting. MAVS aggregation–associated gene expression signature was analyzed by microarray.ResultsIn blood cells from 22 of 67 SLE patients, essentially all MAVS was in a high molecular weight aggregated form. None of 6 rheumatoid arthritis patients and only 3 of 33 healthy controls had abnormal MAVS. Compared to MAVS aggregate–negative patients, MAVS aggregate–positive SLE patients had significantly higher serum levels of IFNβ and significantly increased levels of autoantibodies against Sm and U1 RNP. Gene array data revealed a characteristic gene expression pattern in these patients, with altered expression of genes involved in IFN signaling and membrane trafficking.ConclusionPersistent MAVS aggregates may lead to increased type I IFN production and result in unmitigated signals leading to autoimmunity.

Systemic lupus erythematosus (SLE) is a complex multisystem autoimmune disease, characterized by a spectrum of autoantibodies that target multiple cellular components. Glomerulonephritis is a major cause of morbidity in patients with SLE. Little is known about the pathogenesis of SLE renal damage and compromised renal function. Activation of both Stat1 and Stat3 has been reported in lupus and lupus nephritis. The reciprocal activation of these two transcription factors may have a major impact on renal inflammation. To study the role of Stat1 in a lupus model, we induced lupus-like chronic graft-versus-host disease (cGVHD) in Stat1-knockout (KO) and wild-type (WT) mice by i.p. injection of class II-disparate bm12 splenocytes. WT recipients of these alloreactive cells developed anti-dsDNA autoantibodies starting at week 2 as expected, with a decline after week 4. In contrast, Stat1-KO hosts exhibited a prolonged and significant increase of anti-dsDNA autoantibody responses compared with WT mice (week 4 to week 8). Increased autoantibody titers were accompanied by increased proteinuria and mortality in the cGVHD host mice lacking Stat1. Further analysis revealed expression and activation of Stat3 in the glomeruli of Stat1-KO host mice but not WT mice with cGVHD. Glomerular Stat3 activity in the Stat1-KO mice was associated with increased IL-6 and IFN-γ secretion and macrophage infiltration. Interactions between Stat1 and Stat3 thus appear to be crucial in determining the severity of lupus-like disease in the cGVHD model.

Lupus nephritis (LN) is one of the most serious manifestations of SLE with high incidence of morbidity and mortality. Opposing effects of activated Stat1 and Stat3 have been described for different diseases. Stat1 activation was reported upregulated in the kidney of lupus-prone mice with nephritis, while inhibition of Stat3 in the kidney attenuated kidney inflammation. The present study was undertaken to investigate the role of Stat1 in an experimental animal model of lupus. We induced bm12-B6 chronic graft-versus-host disease (cGVHD) in Stat1-KO and WT mice. WT mice developed anti-dsDNA autoantibodies starting at week 2 as expected, with a decline noted after week 4. In contrast, Stat1-KO mice exhibited a prolonged and significant increase of anti-dsDNA and anti-chromatin autoantibody responses compared to WT mice (week 4 to week 8). Enhanced IgG1 and IgG2b responses but absent IgG3 response were demonstrated in the sera of Stat1-KO mice as compared to WT mice given the same treatment. To our surprise, Stat1-KO mice developed severe nephritis starting at week 8. Three months after cGVHD-induction, 40% Stat1-KO mice were dead. Immunofluorescent staining of kidney sections from the cGVHD Stat1-KO mice revealed an enhanced glomerular Stat3 expression, which correlated with increased IL-6 expression. The observation that altered expression of Stat3 influences IL-6 expression in the glomeruli in this lupus model suggests possible molecular targets for treatment of LN.

Primary viral infections induce activation of CD8(+) T cells responsible for effective resistance. We sought to characterize the nature of the CD8(+) T cell expansion observed after primary viral infection with influenza. Infection of naive mice with different strains of influenza resulted in the rapid expansion of memory CD8(+) T cells exhibiting a unique bystander phenotype with significant up-regulation of natural killer group 2D (NKG2D), but not CD25, on the CD44(high)CD8(+) T cells, suggesting an antigen non-specific phenotype. We further confirmed the non-specificity of this phenotype on ovalbumin-specific (OT-I) CD8(+) T cells, which are not specific to influenza. These non-specific CD8(+) T cells also displayed increased lytic capabilities and were observed primarily in the lung. Thus, influenza infection was shown to induce a rapid, antigen non-specific memory T cell expansion which is restricted to the specific site of inflammation. In contrast, CD8(+) T cells of a similar phenotype could be observed in other organs following administration of systemic agonistic anti-CD40 and interleukin-2 immunotherapy, demonstrating that bystander expansion in multiple sites is possible depending on whether the nature of activation is either acute or systemic. Finally, intranasal blockade of NKG2D resulted in a significant increase in viral replication early during the course of infection, suggesting that NKG2D is a critical mediator of anti-influenza responses prior to the initiation of adaptive immunity. These results characterize further the local bystander expansion of tissue-resident, memory CD8(+) T cells which, due to their early induction, may play an important NKG2D-mediated, antigen non-specific role during the early stages of viral infection.

Decades of research on mammalian immunity to influenza virus infection have thoroughly established the important contributions made by both the innate and adaptive responses in containing the infection, and in eliminating the virus and protecting from reinfection, respectively. While rapid non-specific innate response is functionally distinct from, yet elegantly complementary to, the delayed-but-specific adaptive response, an increasing number of studies have provided evidence suggesting signals generated during the early innate response can have a significant impact on the quality of the later adaptive response, particularly in the context of influenza virus infection. From these findings emerged the notion that certain innate signals can act directly on B cells, and that this can even help activate virus specific B cells independent of T cell help, marking a major shift away from the current two-signal paradigm of lymphocyte activation. Here we review the current understanding of early B cell responses to influenza virus infection and the role of innate signals (particularly IFN-I and TLR7) in shaping this response.

Complex developmental and immune regulatory programs ensure that B cells respond to foreign but not to self-antigens. However, B cell responses can also be triggered non-specifically by mitogens, such as TLR-agonists, which seems counterintuitive and even dangerous. Furthermore, our studies showed a strong, albeit transient and highly specific increase of TLR3 and TLR7 in B cells that had accumulated in regional lymph nodes within 48h after infection with influenza virus, causing enhanced responsiveness of B cells to TLR7 agonists. This increase was induced by direct stimulation of B cells via the type I IFNR. While these observations provide a mechanism for the observed enhancing effects of type I IFN on antiviral B cell responses, they raise the question of how self-reactive responses are avoided, given that TLR7 overexpression has been directly linked to enhanced self-reactivity and autoimmune diseases. We hypothesize that signals must exist that counterbalance this increased sensitivity of B cells to TLR7 signals during influenza infection. Our data now suggest that type I IFN itself might provide such a counterbalance by suppressing B cell cell-cycle progression. Together our data indicate a dual role for type I IFN in differentially regulating specific and non-specific B cell responses after infection. They also raise questions about the role and function of the increased levels of type I IFN observed in patients with certain autoimmune diseases such as SLE.

Memory CD8 T cells (CD44hi+) have a wide distribution in peripheral blood, secondary lymphoid and non-lymphoid tissues and are hallmarked by their ability to respond rapidly to antigenic re-challenge. We observed that systemic cytokine immunotherapy (IT) results in the marked expansion of memory CD8 T cells due to the preferential expansion of pre-existing memory T cells and not the conversion of naïve (CD44lo+) T cells to an activated phenotype. These CD8 T cells express NKG2D and have cytolytic activity; however, a lack of increased CD25 and PD-1 expression suggests that the expansion and activation are independent of TCR engagement. Studies of CD8 T cells from OT-1 TCR transgenic mice after IT demonstrated the increased lysis of ova-negative tumor targets; moreover, the OT-1 CD8 T cells possessed a memory phenotype and lacked CD25 expression in the absence of ova vaccination. To determine if memory CD8 T cells play a role in pathogenic situations, mice were infected with influenza and tissues were examined for the presence of CD25-NKG2D+ memory CD8 T cells. Interestingly, these cells expanded rapidly in the lungs of infected mice but not peripheral tissues, indicating that memory T cells may play a role in the clearance of pathogen, regardless of antigen specificity. These data suggest that resident tissue memory CD8 T cells may act in reserve as innate effectors and thus bridge the gap between adaptive and innate immunity in cancer immunotherapy and viral infections.

B cell responses are regulated by Ag recognition, costimulatory signals provided by interaction with helper T cells, and by innate signals. We recently provided evidence for a link between the effects of innate and costimulatory signals on B cells during influenza virus infection, by demonstrating that most B cells in the regional lymph nodes of the respiratory tract enhance surface expression of the costimulator B7-2 (CD86) within 24-48 h following infection via a type I IFNR-dependent mechanisms, a finding we are confirming here. While the role of B7-1/2 for helper T cell activation is well documented, its role in direct B cell regulation is poorly understood. Here, our in vivo studies with mixed bone marrow irradiation chimeric mice, lacking B7-1/2 only on B cells, demonstrated that B7-1/2 expression is crucial for induction of maximal local, but to a lesser extent systemic, IgG Ab responses following influenza virus infection. In contrast to mice that completely lack B7-1/2 expression, loss of B7-1/2 on B cells alone did not significantly affect germinal center formation or the extent of CD4(+) T cell activation and IFN-gamma secretion. Instead, our in vitro studies identify a dramatic effect of B7-2 engagement on IgG, but not IgM secretion by already class-switched B cells. Concomitantly, B7-2 engagement induced expression of X-box binding protein 1 (XBP-1) and spliced XBP1, evidence for increased protein synthesis by these cells. Taken together, these results identify direct signaling through B7-1/2 as a potent regulator of IgG secretion by previously activated B cells.

C omplex interactions among cells presenting and recognizing Ags are involved in the initiation and regulation of adaptive immune responses (1). T cell-dependent B cell responses require reciprocal interactions between T and B cells that are dependent on engagement of appropriate BCR complexes, costimulatory molecules, and innate signals (1– 4). Among the most important costimulatory molecules are those involving the B7 family members B7-1 (CD80) and B7-2 (CD86) (3, 5, 6). These receptors are expressed on APCs (dendritic cells, macro-phages, and B cells) and are rapidly up-regulated by inflammatory as well as Ag-specific signals for enhanced interaction with CD28 or CTLA-4 expressed on T cells (3). Whereas costimulatory molecules appear to be required for full B cell activation, the presence of additional " third " signals, that is, innate signals such as TLR agonists (7–9) and/or cytokines such as type I IFN (10 –13), seem to control and regulate the magnitude and quality of the specific B cell responses. We provided evidence for a linkage between the effects of innate and costimulatory signals on B cells during influenza virus infection by demonstrating that most B cells in the regional mediastinal lymph nodes (MedLN) 4 of the respiratory tract enhance surface expression of the costimulator B7-2 within 24 – 48 h following infection. At that time B7-2 induction is dependent entirely on direct type I IFNR-mediated signals to B cells (10, 11). This widespread IFN-driven B7-2 up-regulation is thus one of the first responses of B cells at the local site of infection during early influenza virus infection. Direct type I IFN-mediated B cell activation significantly affects the quality and magnitude of the antiviral humoral response (10 – 13). As we and others showed previously, mice deficient in type I IFNR or lacking the IFNR only on B cells showed reduced virus-specific IgM, IgA, and IgG responses as well as alterations in the isotype profile of those responses that did develop. Specifically, type I IFN affected the isotype profile of the response with a shift in the ratio of IgG2a/IgG1 caused by reduced secretion of IgG2a and enhanced secretion of IgG1 (11). Studies by others have provided solid evidence that B7/CD28-mediated signaling regulates B cell responses. The blockade of CD28-B7-1/2 interactions using CTLA4-Ig treatment causes a reduction in overall antiviral Ab production following influenza virus infection (14). Mice deficient in CD28 or in both B7-1 and B7-2 (B7.1/2 Ϫ/Ϫ) lack germinal …

Innate signaling is an essential part of the anti-viral immune response. One such signal induced during an infection is type-I interferon (IFN), an anti-viral cytokine with immune modulatory effects on many cell types, including B cells. It also affects cell cycling and survival. Other signals are delivered via innate toll-like receptors (TLR) that can induce B cell proliferation in response to select microbial products, independent of, or in addition to B cell receptor signaling. Previous studies showed that these two innate stimuli affect the magnitude and quality of the local influenza virus-specific B cell response, and the sensitivity of B cells to non-specific activation via self-antigens. The precise mechanisms by which these innate TLR and type-I IFN signals exert their effects on the antiviral B cell responses, however, remain unclear. We show that type-I IFN regulates steady-state expression of innate viral recognition receptors TLR3 and TLR7 in B cells and their induction in local B cells following infection with influenza virus. IFN also induced several other factors regulating cell cycling and survival, notably cyclin D2 and Bcl-2, in these cells. Furthermore, stimulation by both TLR7 and type-I IFN, but not TLR3, strongly modulated cell cycle entry and survival of B cells. Our data identify the control of cell cycle and survival as key effects of TLR7 and type-I IFN on B cell responses. Supported by NIH AI51354.