miR‐155 deficiency protects mice from experimental colitis by reducing T helper type 1/type 17 responses

Inflammatory bowel disease (IBD), a chronic intestinal inflammatory condition that affects millions of people worldwide, results in high morbidity and exorbitant health-care costs. The critical features of both innate and adaptive immunity are to control inflammation and dysfunction in this equilibrium is believed to be the reason for the development of IBD. miR-155, a microRNA, is up-regulated in various inflammatory disease states, including IBD, and is a positive regulator of T-cell responses. To date, no reports have defined a function for miR-155 with regard to cellular responses in IBD. Using an acute experimental colitis model, we found that miR-155^−/− mice, as compared to wild-type control mice, have decreased clinical scores, a reversal of colitis-associated pathogenesis, and reduced systemic and mucosal inflammatory cytokines. The increased frequency of CD4⁺ lymphocytes in the spleen and lamina propria with dextran sodium sulphate induction was decreased in miR-155^−/− mice. Similarly, miR-155 deficiency abrogated the increased numbers of interferon-γ expressing CD4⁺ T cells typically observed in wild-type mice in this model. The frequency of systemic and mucosal T helper type 17-, CCR9-expressing CD4⁺ T cells was also reduced in miR-155^−/− mice compared with control mice. These findings strongly support a role for miR-155 in facilitating pro-inflammatory cellular responses in this model of IBD. Loss of miR-155 also results in decreases in T helper type 1/type 17, CD11b^+, and CD11c⁺ cells, which correlated with reduced clinical scores and severity of disease. miR-155 may serve as a potential therapeutic target for the treatment of IBD.     

Regulation of inflammasome activity

Granulomatous experimental autoimmune thyroiditis (G-EAT) is induced by mouse thyroglobulin (MTg)-sensitized splenocytes activated with MTg and interleukin (IL)-12. Our previous studies showed that, when used as donors and recipients, interferon (IFN)-γ^−/− and wild-type (WT) DBA/1 mice both develop severe G-EAT. Thyroid lesions in IFN-γ^−/− mice have many eosinophils and few neutrophils, while those in WT mice have extensive neutrophil infiltration and few eosinophils. Thyroid lesions in IFN-γ^−/− mice consistently resolve by day 40–50, whereas those in WT mice have ongoing inflammation and fibrosis persisting for more than 60 days. To determine if the extensive infiltration of eosinophils in thyroids of IFN-γ^−/− mice contributes to thyroid damage and/or early resolution of G-EAT, anti-IL-5 was used to inhibit migration of eosinophils to thyroids. G-EAT severity was compared at day 20 and day 40–50 in IFN-γ^−/− recipients given anti-IL-5 or control immunoglobulin G (IgG). Thyroids of anti-IL-5-treated IFN-γ^−/− mice had few eosinophils and more neutrophils at day 20, but G-EAT severity scores were comparable to those of control IgG-treated mice at both day 20 and day 40–50. Expression of chemokine (C-X-C motif) ligand 1 (CXCL1) mRNA was higher and that of chemokine (C-C motif) ligand 11 (CCL11) mRNA was lower in thyroids of anti-IL-5-treated IFN-γ^−/− mice. IL-5 neutralization did not influence mRNA expression of most cytokines in IFN-γ^−/− mice. Thus, inhibiting eosinophil migration to thyroids did not affect G-EAT severity or resolution in IFN-γ^−/− mice, suggesting that eosinophil infiltration of thyroids occurs as a consequence of IFN-γ deficiency, but these cells have no apparent pathogenic role in G-EAT.     

MicroRNA155 Plays a Critical Role in the Pathogenesis of Cutaneous Leishmania major Infection by Promoting a Th2 Response and Attenuating Dendritic Cell Activity

Interferon (IFN)-γ is indispensable in the resolution of cutaneous leishmaniasis (CL), while the Th2 cytokines IL-4, IL-10, and IL-13 mediate susceptibility. A recent study found that miR155, which promotes CD4⁺ Th1 response and IFN-γ production, is dispensable in the control of Leishmania donovani infection. Here, the role of miR155 in CL caused by L. major was investigated using miR155-deficient (miR155^−/−) mice. Infection was controlled significantly quicker in the miR155^−/− mice than in their wild-type (WT) counterparts, indicating that miR155 contributes to the pathogenesis of CL. Faster resolution of infection in miR155^−/− mice was associated with increased levels of Th1-associated IL-12 and IFN-γ and reduced production of Th2- associated IL-4, IL-10, and IL-13. Concentrations of IFN-γ⁺CD8⁺ T cells and natural killer cells in draining lymph nodes were significantly higher in the L. major−infected miR155^−/− mice than in the infected WT mice, as indicated by flow-cytometry. After in vitro IFN-γ stimulation, nitric oxide and IL-12 production were increased, IL-10 production was decreased, and parasite clearance was enhanced in L. major−infected miR155^−/− DCs compared to those in WT DCs. Furthermore, IFN-γ production from activated miR155^−/− T cells was significantly enhanced in L. major−infected miR155^−/− DCs. Together, these findings demonstrate that miR155 promotes susceptibility to CL caused by L. major by promoting Th2 response and inhibiting DC function.

Leishmania are obligate intracellular protozoans that infect phagocytes and cause a spectrum of clinical diseases such as cutaneous leishmaniasis (CL) and visceral leishmaniasis. Common in the tropical and subtropical regions, leishmaniasis affects over 1 billion people worldwide, with an incidence of up to 1 million cases per year.¹ CL is the most common type of Leishmania infection, manifesting as localized skin lesions that can become chronic, leading to significant tissue destruction and disfigurement.^2,3 It is well documented that the induction of a Th1 response and interferon (IFN)-γ are indispensable in the resolution of CL caused by Leishmania major,⁴ whereas disease progression is associated with the induction of a Th2 response and the production of cytokines such as IL-4 and IL-10.⁵ Establishing a disease-resolving response in the host is largely dependent on the ability to mount an appropriate Th1 immune response.⁴ Crucial in this response is the stimulation and activation of DCs that direct T-cell proliferation and differentiation toward IFN-γ–producing Th1 cells.^6,7 In addition to activating of phagocytic cells, IFN-γ induces the production of reactive nitrogen species, specifically nitric oxide (NO), leading to enhanced parasite clearance.⁴miR155 is a recognized regulator of immune cell function and immune response. miR155 enhances macrophage and DC activation and induces inflammatory response,^8,9 and up-regulation of miR155 in CD4⁺ T cells promotes preferential Th1 differentiation and IFN-γ production¹⁰ by suppressing the expression of suppressor of cytokine signaling (SOCS)-1.11, 12, 13, 14 Conversely, miR155 gene–deficient mice exhibit diminished levels of Th1/Th17 cells, macrophages, and DCs.¹⁵ miR155 has also been shown to play a role in regulating effector Th2 response.16, 17, 18 Collectively, these findings suggest that miR155 regulates both Th1 and Th2 responses, which control the outcome of CL caused by L. major. Therefore, the role of miR155 in immunity to L. major using miR155^−/− mice was investigated in the present study. The findings show that miR155 is not required for the induction of a Th1 response and IFN-γ in L. major infection. Rather, miR155 plays a disease-exacerbating role in CL by attenuating DC function and Th1 response and promoting Th2 response.     

Calcineurin deficiency decreases inflammatory lesions in transforming growth factor β1‐deficient mice

Transforming growth factor (TGF) β1) is an immunoregulatory cytokine involved in self-tolerance and lymphocyte homeostasis. Tgfb1 knock-out (KO) mice develop severe multi-focal autoimmune inflammatory lesions due to [Ca²⁺]i deregulation in T cells, and die within 3 weeks after birth. Because the calcineurin inhibitor FK506 inhibits the hyperresponsiveness of Tgfb1^−/− thymocytes, and because calcineurin Aβ (CNAβ)-deficient mice do not reject allogenic tumours, we have generated Tgfb1^−/−Cnab^−/− mice to address whether CNAβ deficiency prevents T cell activation and inflammation in Tgfb1^−/− mice. Here we show that in Tgfb1^−/−Cnab^−/− mice inflammation is reduced significantly relative to that in Tgfb1^−/− mice. However, both CD4⁺ and CD8⁺ T cells in double knock-out (DKO) mice are activated, as revealed by up-regulation of CD11a lymphocyte function-associated antigen-1 (LFA-1), CD44 and CD69 and down-regulation of CD62L. These data suggest that deficiency of CNAβ decreases inflammatory lesions but does not prevent activation of autoreactive T cells. Also Tgfb1^−/− T cells can undergo activation in the absence of CNAβ, probably by using the other isoform of calcineurin (CNAα) in a compensatory manner. CNAβ-deficient T cells undergo spontaneous activation in vivo and are activated upon anti-T cell receptor stimulation in vitro. Understanding the role of calcineurin in T cell regulation should open up new therapeutic opportunities for inflammation and cancer.     

Interferon‐γ‐ and glucocorticoid‐mediated pathways synergize to enhance death of CD4+ CD8+ thymocytes during Salmonella enterica serovar Typhimurium infection

Thymic atrophy is known to occur during infections; however, there is limited understanding of its causes and of the cross-talk between different pathways. This study investigates mechanisms involved in thymic atrophy during a model of oral infection by Salmonella enterica serovar Typhimurium (S. typhimurium). Significant death of CD4⁺ CD8⁺ thymocytes, but not of single-positive thymocytes or peripheral lymphocytes, is observed at later stages during infection with live, but not heat-killed, bacteria. The death of CD4⁺ CD8⁺ thymocytes is Fas-independent as shown by infection studies with lpr mice. However, apoptosis occurs with lowering of mitochondrial potential and higher caspase-3 activity. The amounts of cortisol, a glucocorticoid, and interferon-γ (IFN-γ), an inflammatory cytokine, increase upon infection. To investigate the functional roles of these molecules, studies were performed using Ifnγ^−/− mice together with RU486, a glucocorticoid receptor antagonist. Treatment of C57BL/6 mice with RU486 does not affect colony-forming units (CFU), amounts of IFN-γ and mouse survival; however, there is partial rescue in thymocyte death. Upon infection, Ifnγ^−/− mice display higher CFU and lower survival but more surviving thymocytes are recovered. However, there is no difference in cortisol amounts in C57BL/6 and Ifnγ^−/− mice. Importantly, the number of CD4⁺ CD8⁺ thymocytes is significantly higher in Ifnγ^−/− mice treated with RU486 along with lower caspase-3 activity and mitochondrial damage. Hence, endogenous glucocorticoid and IFN-γ-mediated pathways are parallel but synergize in an additive manner to induce death of CD4⁺ CD8⁺ thymocytes during S. typhimurium infection. The implications of this study for host responses during infection are discussed.     

A serotype 3 pneumococcal capsular polysaccharide-specific monoclonal antibody requires Fcγ receptor III and macrophages to mediate protection against pneumococcal pneumonia in mice

Antibodies to pneumococcal capsular polysaccharide (PPS) are required for PPS-based vaccine-mediated protection against Streptococcus pneumoniae. Previous work established that 1E2, a mouse IgG1 to PPS3 that does not induce serotype 3 (ST3) S. pneumoniae killing by phagocytes in vitro, protects mice from death after intranasal infection with ST3, but its efficacy was abrogated in FcγR (F common gamma receptor)-deficient mice. In this study, we determined whether 1E2 efficacy against pulmonary ST3 infection requires FcγRIII. 1E2 did not protect FcγRIII-deficient (FcγRIII^−/−) mice. Studies of the mechanism of 1E2-mediated effects showed that it resulted in a marked reduction in lung inflammation in ST3-infected wild-type (Wt [C57BL/6]) mice that was abrogated in FcγRIII^−/− mice. 1E2 had no effect on early bacterial clearance in the lungs of ST3-infected Wt, FcγRIIB^−/−, or FcγRIII^−/− mice, but it reduced levels of bacteremia and serum macrophage inflammatory protein-2) (MIP-2), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF-α) in Wt and FcγRIIB^−/− mice, strains in which it is protective. As previous work showed that neutrophils were dispensable for 1E2 efficacy, we investigated whether macrophages are required for 1E2 efficacy against intranasal infection with ST3 and found that its efficacy was abrogated in Wt mice depleted of macrophages intranasally. In vitro studies revealed that1E2 promoted ST3 internalization by naïve alveolar macrophages but did not induce early intracellular killing. Macrophages from 1E2-treated ST3-infected mice studied ex vivo exhibited more apoptosis than those from FcγRIII^−/− mice. These findings suggest that 1E2 mediates protection against ST3 in mice by affecting the inflammatory response, perhaps in part via macrophage apoptosis, rather than by inducing early bacterial clearance.     

Toll‐like receptor 4 signalling through MyD88 is essential to control Salmonella enterica serovar Typhimurium infection,but not for the initiation of bacterial clearance

Toll-like receptor-4 (TLR4) is important in protection against lethal Salmonella enterica serovar Typhimurium (S. Typhimurium) infection. Control of the early stages of sublethal S. Typhimurium infection in mice depends on TLR4-dependent activation of macrophages and natural killer (NK) cells to drive an inflammatory response. TLR4 signals through the adapter proteins Mal/MyD88 and TRIF-related adaptor molecule (TRAM)/TIR-domain-containing adaptor-inducing interferon-b (TRIF). In the mouse typhoid model we showed that TLR4 and MyD88, but not Mal or TRIF, are essential for the control of exponential S. Typhimurium growth. TRIF^−/− mice have a higher bacterial load in comparison with wild-type mice during a sublethal infection because TRIF is important for bacterial killing during the first day of systemic disease. Minimal pro-inflammatory responses were induced by S. Typhimurium infection of macrophages from TLR4^−/−, MyD88^−/− and TRIF^−/− mice in vitro. Pro-inflammatory responses from Mal^−/− macrophages were similar to those from wild-type cells. The pro-inflammatory responses of TRIF^−/− macrophages were partially restored by the addition of interferon-γ (IFN-γ), and TRIF^−/− mice produced markedly enhanced IFN-γ levels, in comparison to wild-type mice, probably explaining why bacterial growth can be controlled in these mice. TLR4^−/−, MyD88^−/−, TRIF^−/− and Mal^−/− mice all initiated clearance of S. Typhimurium, suggesting that TLR4 signalling is not important in driving bacterial clearance in comparison to its critical role in controlling early bacterial growth in mouse typhoid.     

β-Arrestin-1 Deficiency Protects Mice from Experimental Colitis

β-Arrestins are intracellular scaffolding proteins that modulate specific cell signaling pathways. Recent studies, in both cell culture and in vivo models, have demonstrated an important role for β-arrestin-1 in inflammation. However, the role of β-arrestin-1 in the pathogenesis of inflammatory bowel disease (IBD) is not known. Our goal was to investigate the role of β-arrestin-1 in IBD using mouse models of colitis. To this end, we subjected wild-type (WT) and β-arrestin-1 knockout (β-arr-1^−/−) mice to colitis induced by trinitrobenzenesulfonic acid or dextran sulfate sodium and examined the clinical signs, gross pathology, and histopathology of the colon, as well as inflammatory components. The β-arr-1^−/− mice displayed significantly attenuated colitis, compared with WT mice, in both models. Consistent with the phenotypic observations, histological examination of the colon revealed attenuated disease pathology in the β-arr-1^−/− mice. Our results further demonstrate that β-arr-1^−/− mice are deficient in IL-6 expression in the colon, but have higher expression of the anti-inflammatory IL-10 family of cytokines. Our results also demonstrate diminished ERK and NFκB pathways in the colons of β-arr-1^−/− mice, compared with WT mice. Taken together, our results demonstrate that decreased IL-6 production and enhanced IL-10 and IL-22 production in β-arrestin-1–deficient mice likely lead to attenuated gut inflammation.Arrestins are scaffolding proteins now classified into α and β arrestin families.¹ The β-arrestins were discovered for their role in binding to phosphorylated G-protein coupled receptors (GPCRs) to evoke receptor desensitization. Of the four members of the β-arrestin family, β-arrestin-1 (originally arrestin-2) and β-arrestin-2 (originally arrestin-3) are ubiquitously distributed. Even though their role in receptor desensitization has been well characterized and has been shown to have pharmacological and therapeutic implications, recent studies indicate that β-arrestins have a much broader role in cell signaling related to both GPCRs and non-GPCRs.² In addition, although several members of the arrestin family are present during development, the crucial ones are β-arrestins 1 and 2, because deletion of both results in embryonic lethality.³ Several studies have shown that β-arrestins, by virtue of regulating cell signaling, are able to modulate a variety of cell biological processes including gene expression, chemotaxis, proliferation, and apoptosis. Although β-arrestin-1 and -2 have been shown to have many overlapping cellular functions, they also have unique roles of their own.²Because of their critical role in many cellular functions, β-arrestins are crucial in the pathogenesis of many different diseases, including Parkinson''s disease, multiple sclerosis, cardiovascular disease, rheumatoid arthritis, sepsis, and allergic asthma.^4–8 In this context, we recently showed that β-arrestin-1 and -2 have differential and overlapping roles in endotoxin- and adenovirus-induced inflammatory responses in vivo.^9,10 In addition, other researchers have also shown that β-arrestin-2 plays a crucial role in sepsis and arthritis models of inflammatory disease.^7,11 Although it is clear that β-arrestins are important modulators of inflammation, nonetheless the role of β-arrestins in gastrointestinal inflammation is not known.Inflammatory bowel disease (IBD) affects more than 1.4 million people in the United States and accounts for more than $1.7 billion dollars in health care costs.¹² IBD broadly includes both Crohn''s disease and ulcerative colitis, both of which are characterized by chronic relapsing inflammation of the gastrointestinal tract.¹³ Importantly, IBD requires lifetime care and currently has no medical cure. Understanding the mechanisms involved in the pathogenesis of IBD is crucial for developing new therapeutic strategies to prevent or cure this inflammatory disease. In this regard, recent studies have shown that activation of Toll-like receptors (TLRs) and production of inflammatory cytokines are important mediators in the pathogenesis of colitis, including in mouse models of chemically induced colitis.^14–17 Previous studies from our laboratory have demonstrated that β-arrestin-1 mediates TLR signaling and the consequent inflammatory cytokine production in vivo.⁹ Based on these data, we hypothesized that β-arrestin-1 is an important and critical regulator of colitis pathogenesis. Using two different colitis models, we demonstrate here that β-arrestin-1 is a crucial mediator of colitis pathogenesis in mice. Deficiency of β-arrestin-1 almost completely abrogated the development of intestinal inflammation in the dextran sulfate sodium (DSS) model of colitis, whereas inflammation was significantly attenuated in the trinitrobenzenesulfonic acid (TNBS) model. Furthermore, our results also demonstrate that lack of β-arrestin-1 leads to diminished production of IL-6 but enhanced production of cytokines IL-10 and IL-22, and in combination these likely result in a favorable outcome for intestinal inflammation. The present study implicates a β-arrestin-1–mediated signaling pathway as a potential molecular target in the treatment of colitis.     

Antigen‐triggered interferon‐γ and interleukin‐10 pattern in cured mucosal leishmaniasis patients is shaped during the active phase of disease

An exacerbated type 1 response to leishmanial antigens is the basis of tissue destruction observed in mucosal leishmaniasis (ML). After therapy, a persistent production of high levels of inflammatory cytokines can confer a poor prognosis. Herein we investigated whether the clinical conditions defined during the active phase of ML affect the magnitude of long-term anti-Leishmania immune response. Twenty clinically cured ML cases were studied. Peripheral blood mononuclear cells (PBMC) were cultured with L. braziliensis antigens (Lb-Ag), Toxoplasma gondii antigens (Tg-Ag), concanavalin-A (Con-A) or medium alone, and the lymphocyte proliferative response and cytokine secretion were quantified. Medical records were reviewed for Montenegro skin test (MST) during diagnosis, duration of ML disease or time elapsed after clinical cure. The duration of disease was correlated positively with MST (r = 0·61). Lb-Ag induced interferon (IFN)-γ was correlated positively with duration of illness (r = 0·69) as well as the frequency of secreting cells [enzyme-linked immunospot (ELISPOT)] assay. No association was observed for Tg-Ag or Con-A. Disease duration was correlated negatively with interleukin (IL)-10 production (r = −0·76). Moreover, a negative correlation between length of time after clinical cure and TNF levels (r = −0·94) or the IFN-γ : IL-10 ratio (r = −0·89) were also seen. We suggest that the magnitude of the IFN-γ inflammatory response triggered by ML can be driven by the time of leishmanial antigens exposition during the active phase of the disease. This pattern could persist even long-term after cure. However, despite IFN-γ levels, the decrease of the TNF and IFN-γ : IL-10 ratio reflects the control of proinflammatory responses achieved by cure of ML, possibly preventing disease relapses.     

MyD88 signaling contributes to early pulmonary responses to Aspergillus fumigatus

Toll-like receptors and the β-glucan receptor, dectin-1, mediate macrophage inflammatory responses to Aspergillus fumigatus through MyD88-dependent and -independent signaling mechanisms; however, pulmonary inflammatory responses in MyD88-deficient mice challenged with A. fumigatus are poorly defined. The role of MyD88 signaling in early pulmonary inflammation and fungal clearance was evaluated in C57BL/6J wild-type (WT) and MyD88-deficient (MyD88^−/−) mice. Early (<48 h) after infection, MyD88^−/− mice had higher fungal burdens than those of WT mice, although fungal burdens rapidly declined (>72 h) in both. MyD88^−/− mice had less consolidated inflammation, with fewer NK cells, in lung tissue early (24 h) after infection than did WT mice. At the latter time point, MyD88^−/− mouse lungs were characterized by a large amount of necrotic cellular debris and fibrin, while WT lungs had organized inflammation. Although there were equivalent numbers of macrophages in WT and MyD88^−/− mouse lung tissues, MyD88^−/− cells demonstrated delayed uptake of green fluorescent protein-expressing A. fumigatus (GFP-Af293); histologically, MyD88^−/− mouse lungs had more hyphal invasion of terminal airways and vessels, the appearance of bronchiolar epithelial cell necrosis, and necrotizing vasculitis. MyD88^−/− lung homogenates contained comparatively decreased amounts of interleukin-1β (IL-1β), IL-6, KC, and gamma interferon and paradoxically increased amounts of tumor necrosis factor alpha and macrophage inflammatory protein 1α. These data indicate that the MyD88-dependent pathway mediates acute pulmonary fungal clearance, inflammation, and tissue injury very early after infection. Resolution of abnormalities within a 3-day window demonstrates the importance of redundant signaling pathways in mediating pulmonary inflammatory responses to fungi.     

Role of miR-155 in the Pathogenesis of Herpetic Stromal Keratitis

Ocular infection with herpes simplex virus 1 can result in a chronic immunoinflammatory stromal keratitis (SK) lesion that is a significant cause of human blindness. A key to controlling SK lesion severity is to identify cellular and molecular events responsible for tissue damage and to manipulate them therapeutically. Potential targets for therapy are miRNAs, but these are minimally explored especially in responses to infection. Here, we demonstrated that Mir155 expression was up-regulated after ocular herpes simplex virus 1 infection, with the increased Mir155 expression occurring mainly in macrophages and CD4⁺ T cells and to a lesser extent in neutrophils. In vivo studies indicated that Mir155 knockout mice were more resistant to herpes SK with marked suppression of T helper cells type 1 and 17 responses both in the ocular lesions and the lymphoid organs. The reduced SK lesion severity was reflected by increased phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 and interferon-γ receptor α-chain levels in activated CD4⁺ T cells in the lymph nodes. Finally, in vivo silencing of miR-155 by the provision of antagomir-155 nanoparticles to herpes simplex virus 1–infected mice led to diminished SK lesions and corneal vascularization. In conclusion, our results indicate that miR-155 contributes to the pathogenesis of SK and represents a promising target to control SK severity.Ocular infection with herpes simplex virus 1 (HSV-1) can result in a chronic tissue-damaging response in the stroma, which is considered to be largely the consequence of a host inflammatory response to the infection.¹ This concept is strongly supported by animal model studies in which lesions were shown to be mainly orchestrated by CD4⁺ T cells with neutrophils and macrophage largely responsible for the tissue damage.^2–5 Several effective control measures for stromal keratitis (SK) are suggested.⁶ These include approaches that influence cellular infiltration and activation of the proinflammatory mediators of SK.⁶ One potential means of modulating SK lesions that so far has received minimal attention is to manipulate the expression of miRNA species that affect either virus or host events during SK.A prime miRNA candidate for consideration is miR-155 because this miRNA can influence the expression of several immune events that contribute to tissue damage.^7–10 For example, animals unable to produce miR-155 because of gene knockout may develop milder lesions in some models of autoimmunity,^8,11–13 and suppressing miR-155 expression, as can be achieved by treatment with antagomirs, holds promise as a means of therapy for autoimmunity.¹³ However, the absence of miR-155 can result in higher susceptibility to some virus infections and some tumors in part because protective CD8⁺ T-cell responses are diminished.^14–17 In fact, overexpression of miRNA can result in enhanced CD8⁺ T-cell–mediated immune protection with some tumors.¹⁷Few studies have focused on the role of miR-155 in situations in which the immune response to an infectious agent may be a principal cause of tissue damage. This is the situation in ocular lesions of the cornea after HSV-1 infection. Here, we have compared the disease outcome after HSV-1 infection in miRNA-155 knockout (Mir155^−/−) mice and in mice in which miR-155 was suppressed by antagomir therapy with controls. We show that HSV-1 infection resulted in up-regulation of miR-155, which was mainly produced by the inflammatory cells and T cells in the cornea. Suppression of miR-155 production resulted in milder lesions that were associated with diminished responses of T helper cells type 1 (Th1) and type 17 (Th17) and reduced inflammatory cytokine production. We also demonstrated that miR-155 suppression resulted in increased phosphatidylinositol-3,4,5-trisphosphate 5-phosphatase 1 (Ship1) and IFN-γ receptor α-chain (IFN-γRα) levels, molecules known to be required for IFN-γ expression and Th1 differentiation.^17,18 These results indicate that miR-155 regulates differentiation and effector function of Th1 cells. Thus, our results suggest that miR-155 could be a promising therapeutic target to treat SK.     

Deletion of Mir155 Prevents Fas-Induced Liver Injury through Up-Regulation of Mcl-1

Fas-induced apoptosis is involved in diverse liver diseases. Herein, we investigated the effect of Mir155 deletion on Fas-induced liver injury. Wild-type (WT) mice and Mir155 knockout (KO) mice were i.p. administered with the anti-Fas antibody (Jo2) to determine animal survival and the extent of liver injury. After Jo2 injection, the Mir155 KO mice exhibited prolonged survival versus the WT mice (P < 0.01). The Mir155 KO mice showed lower alanine aminotransferase and aspartate aminotransferase levels, less liver tissue damage, fewer apoptotic hepatocytes, and lower liver tissue caspase 3/7, 8, and 9 activities compared with the WT mice, indicating that Mir155 deletion prevents Fas-induced hepatocyte apoptosis and liver injury. Hepatocytes isolated from Mir155 KO mice also showed resistance to Fas-induced apoptosis, in vitro. Higher protein level of myeloid cell leukemia-1 (Mcl-1) was also observed in Mir155 KO hepatocytes compared to WT hepatocytes. A miR-155 binding site was identified in the 3′-untranslated region of Mcl-1 mRNA; Mcl1 was identified as a direct target of miR-155 in hepatocytes. Consistently, pretreatment with a siRNA specific for Mcl1 reversed Mir155 deletion–mediated protection against Jo2-induced liver tissue damage. Finally, restoration of Mir155 expression in Mir155 KO mice abolished the protection against Fas-induced hepatocyte apoptosis. Taken together, these findings demonstrate that deletion of Mir155 prevents Fas-induced hepatocyte apoptosis and liver injury through the up-regulation of Mcl1.miRNAs are a class of small noncoding RNAs that regulate gene expression through binding to specific sequences of their targeted mRNAs.¹ miR-155 was initially identified as a specific miRNA for hematopoietic and immune cells and is among the first miRNAs linked to immunity and inflammation.^2–4 Indeed, miR-155 has been shown to modulate the production of cytokines in various immune cells; the expression of Mir155 is up-regulated in multiple immune cell lineages on stimulation with Toll-like receptor ligands, inflammatory cytokines, and specific antigens.^5–9 Subsequent studies have shown that miR-155 also mediates functions outside the hematopoietic and immune systems.^10,11 In the liver, miR-155 has been shown to play a role in hepatocarcinogenesis,^12–15 although its mechanism of action remains to be further defined. miR-155 has also been shown to contribute to alcohol-induced liver injury through induction of tumor necrosis factor α production in macrophages.¹⁶ Interestingly, the level of miR-155 is increased in serum and plasma in patients with alcoholic and inflammatory liver injuries.^17,18 These observations suggest a potential role of miR-155 in liver injury and liver diseases. However, at present, the biological functions and mechanisms of miR-155 in liver cells have not been delineated.The current study aimed to determine the effect and mechanism of miR-155 in Fas and lipopolysaccharide (LPS)/d-galactosamine (D-GalN)–mediated liver injury in mice. Our data show that deletion of Mir155 protects against Fas-induced hepatocyte apoptosis and liver injury but not LPS/D-GalN–induced liver injury. The role of miR-155 in hepatocytes was demonstrated by in vitro studies using hepatocytes isolated from Mir155 knockout (KO) mice. Myeloid cell leukemia-1 (Mcl-1) was identified as a direct target of miR-155 in hepatocytes. Our results reveal a novel role of miR-155 in hepatocytes for regulation of Mcl1 and protection against Fas-induced apoptosis.     

Dietary gluten alters the balance of pro‐inflammatory and anti‐inflammatory cytokines in T cells of BALB/c mice

Several studies have documented that dietary modifications influence the development of type 1 diabetes. However, little is known about the interplay of dietary components and the penetration of diabetes incidence. In this study we tested if wheat gluten is able to induce differences in the cytokine pattern of Foxp3⁺ regulatory T cells, as well as Foxp3⁻ T cells, isolated from intestinal mucosal lymphoid tissue and non-mucosal lymphoid compartments in BALB/c mice. The gluten-containing standard diet markedly changed the cytokine expression within Foxp3⁻ T cells, in all lymphoid organs tested, towards a higher expression of pro-inflammatory interferon-γ (IFN-γ), interleukin-17 (IL-17) and IL-2. In Foxp3⁺ regulatory T cells, gluten ingestion resulted in a mucosal increase in IL-17 and IL-2 and an overall increase in IFN-γ and IL-4. The gluten-free diet induced an anti-inflammatory cytokine profile with higher proportion of transforming growth factor-β (TGF-β)⁺ Foxp3⁻ T cells in all tested lymphoid tissues and higher IL-10 expression within non-T cells in spleen, and a tendency towards a mucosal increase in TGF-β⁺ Foxp3⁺ regulatory T cells. Our data shows that the gluten-containing standard diet modifies the cytokine pattern of both Foxp3⁻ T cells and Foxp3⁺ regulatory T cells towards a more inflammatory cytokine profile. This immune profile may contribute to the higher type 1 diabetes incidence associated with gluten intake.     

Enhanced differentiation of intraepithelial lymphocytes in the intestine of polymeric immunoglobulin receptor‐deficient mice

To clarify the effect of secretory IgA (sIgA) deficiency on gut homeostasis, we examined intraepithelial lymphocytes (IELs) in the small intestine (SI) of polymeric immunoglobulin receptor-deficient (pIgR^−/−) mice. The pIgR^−/− mice exhibited the accumulation of CD8αβ⁺ T-cell receptor (TCR)-αβ⁺ IELs (CD8αβ⁺αβ-IELs) after weaning, but no increase of CD8αβ⁺γδ-IELs was detected in pIgR^−/− TCR-β^−/− mice compared with pIgR^+/+ TCR-β^−/− mice. When 5-bromo-2′-deoxyuridine (BrdU) was given for 14 days, the proportion of BrdU-labelled cells in SI-IELs was not different between pIgR^+/+ mice and pIgR^−/− mice. However, the proportion of BrdU-labelled CD8αβ⁺-IELs became higher in pIgR^−/− mice than pIgR^+/+ mice 10 days after discontinuing BrdU-labelling. Intravenously transferred splenic T cells migrated into the intraepithelial compartments of pIgR^+/+ TCR-β^−/− mice and pIgR^−/− TCR-β^−/− mice to a similar extent. In contrast, in the case of injection of immature bone marrow cells, CD8αβ⁺αβ-IELs increased much more in the SI of pIgR^−/− TCR-β^−/− mice than pIgR^+/+ TCR-β^−/− mice 8 weeks after the transfer. αβ-IELs from pIgR^−/− mice could produce more interferon-γ and interleukin-17 than those of pIgR^+/+ mice, and intestinal permeability tended to increase in the SI of pIgR^−/− mice with aging. Taken together, these results indicate that activated CD8αβ⁺αβ-IELs preferentially accumulate in pIgR^−/− mice through the enhanced differentiation of immature haematopoietic precursor cells, which may subsequently result in the disruption of epithelial integrity.     

Decay-accelerating factor 1 (Daf1) deficiency exacerbates xenobiotic-induced autoimmunity

Absence of decay-accelerating factor 1 (Daf1) has been shown to enhance T-cell responses and autoimmunity via increased expression of specific cytokines, most notably interferon (IFN)-γ. To determine if Daf1 deficiency can exacerbate IFN-γ-dependent murine mercury-induced autoimmunity (mHgIA), C57/BL6 Daf1^+/+ and Daf1^−/− mice were exposed to mercuric chloride (HgCl₂) and examined for differences in cytokine expression, T-cell activation and features of humoral autoimmunity. In the absence of Daf1, mHgIA was exacerbated, with increased serum immunoglobulin G (IgG), anti-nuclear autoantibodies (ANAs) and anti-chromatin autoantibodies. This aggravated response could not be explained by increased T-cell activation but was associated with increased levels of IFN-γ, interleukin (IL)-2, IL-4 and IL-10 but not IL-17 in Daf1-deficient mice. Anti-CD3/anti-CD28 costimulation of Daf1^−/− CD4⁺ T cells in vitro was also found to increase cytokine expression, but the profile was different from that of mHgIA, suggesting that the cytokine changes observed in Daf1 deficiency reflect a response to mercury. The role of Daf1 in influencing cytokine expression was further examined by stimulation of CD4⁺ T cells in the presence of anti-CD3 and CD97, a molecular partner for Daf1. This resulted in increased IL-10, decreased IL-17 and IL-21 and decreased IFN-γ. These findings demonstrate that the absence of Daf1 exacerbates mHgIA, with changes in the profile of expressed cytokines. Interaction between Daf1 and its molecular partner CD97 was found to modify expression of mHgIA-promoting cytokines, suggesting a possible approach for the suppression of overaggressive cytokine production in autoimmunity.