Loss of Sprouty4 in T cells ameliorates experimental autoimmune encephalomyelitis in mice by negatively regulating IL-1β receptor expression

Th17 cells, which have been implicated in autoimmune diseases, require IL-6 and TGF-β for early differentiation. To gain pathogenicity, however, Th17 cells require IL-1β and IL-23. The underlying mechanism by which these confer pathogenicity is not well understood. Here we show that Sprouty4, an inhibitor of the PLCγ-ERK pathway, critically regulates inflammatory Th17 (iTh17) cell differentiation. Sprouty4-deficient mice, as well as mice adoptively transferred with Sprouty4-deficient T cells, were resistant to experimental autoimmune encephalitis (EAE) and showed decreased Th17 cell generation in vivo. In vitro, Sprouty4 deficiency did not severely affect TGF-β/IL-6-induced Th17 cell generation but strongly impaired Th17 differentiation induced by IL-1/IL-6/IL-23. Analysis of Th17-related gene expression revealed that Sprouty4-deficient Th17 cells expressed lower levels of IL-1R1 and IL-23R, while RORγt levels were similar. Consistently, overexpression of Sprouty4 or pharmacological inhibition of ERK upregulated IL-1R1 expression in primary T cells. Thus, Sprouty4 and ERK play a critical role in developing iTh17 cells in Th17 cell-driven autoimmune diseases.     

Regulatory effects of IFN-β on the development of experimental autoimmune uveoretinitis in B10RIII mice

Background

Experimental autoimmune uveoretinitis (EAU) serves as a model for human intraocular inflammation. IFN-β has been used in the treatment of certain autoimmune diseases. Earlier studies showed that it ameliorated EAU; however, the mechanisms involved in this inhibition are still largely unknown.

Methodology/Principal Findings

B10RIII mice were immunized with interphotoreceptor retinoid-binding protein (IRBP) peptide 161–180 in Complete Freund''s adjuvant. Splenocytes from different time points after immunization were used to evaluate the expression of IFN-β. An increased expression of IFN-β was observed during EAU and its highest expression was observed on day 16, 3 days after the peak of intraocular inflammation. Splenocytes and draining lymph node cells from mice immunized with IRBP_161-180 on day 13 and control mice were activated with anti-CD3/anti-CD28 antibodies or IRBP_161-180 to evaluate the production of IFN-γ and IL-17. The results showed that IFN-γ and IL-17 were significantly higher in immunized mice as compared to the control mice when exposed to anti-CD3/anti-CD28 antibodies. However, the production of IFN-γ and IL-17 was detected only in immunized mice, but not in the control mice when stimulated with IRBP_161-180. Multiple subcutaneous injections of IFN-β significantly inhibited EAU activity in association with a down-regulated expression of IFN-γ, IL-17 and an enhanced IL-10 production. In an in vitro system using cells from mice, IFN-β suppressed IFN-γ production by CD4⁺CD62L⁻ T cells, IL-17 production by CD4⁺CD62L^+/- T cells and proliferation of CD4⁺CD62L^+/- T cells. IFN-β inhibited the secretion of IL-6, but promoted the secretion of IL-10 by monocytes. IFN-β-treated monocytes inhibited IL-17 secretion by CD4⁺CD62L^+/- T cells, but did not influence IFN-γ expression and T cell proliferation.

Conclusions/Significance

IFN-β may exert its inhibitory effect on EAU by inhibiting Th1, Th17 cells and modulating relevant cytokines. IFN-β may provide a potential treatment for diseases mediated by Th1 and Th17 cells.     

β-Sitosterol improves experimental colitis in mice with a target against pathogenic bacteria

In this article, we aim to examine the novel effects of β-sitosterol on murine experimental colitis. β-Sitosterol significantly reduces the weight loss, colon length, and alleviated microscopic appearances of colitis induced by dextran sulfate sodium. This compound also decreases the levels of TNF-α, IL-6, and IL-1β in intestinal tissue of mice with experimental colitis in a concentration-dependent manner. β-Sitosterol treatment to intestinal epithelial cells significantly increases expression of antimicrobial peptides and reduces survival of intracellular Salmonella typhimurium. These results showed the multiple effects of β-sitosterol against pathogenic bacteria for a novel approach to the treatment of colonic inflammation.     

Induction of the synthesis of the C-X-C chemokine interferon-γ-inducible protein-10 in experimental canine endotoxemia

Endotoxemia is associated with a systemic inflammatory response leading to organ-specific leukocyte recruitment and tissue injury. Chemokine expression has been demonstrated in various models of sepsis and may mediate tissue infiltration with inflammatory cells. In this study we examined expression of the C-X-C chemokine interferon-gamma-inducible protein-10 (IP-10), a potent T-lymphocyte chemoattractant, in a canine model of endotoxemia and investigated mechanisms of cytokine-mediated IP-10 induction in endothelial cells. Control canine tissues showed negligible expression of IP-10 message, with the exception of the spleen. Endotoxemic dogs demonstrated a robust induction of IP-10 mRNA in the heart, lung, kidney, liver, and spleen. Immunohistochemical studies indicated that IP-10 was predominantly localized in cardiac venular endothelial cells, bronchial epithelial cells, renal mesangial cells, and in the splenic red pulp of endotoxemic dogs. In addition, IP-10 expression was associated with T-lymphocyte infiltration in canine tissues. Tumor necrosis factor-alpha (TNF-alpha) and interleukin-1 beta (IL-1 beta) induced a marked upregulation of IP-10 message in canine venular endothelial cells. IP-10 expression in TNF-alpha-stimulated endothelial cells peaked at 6 h of stimulation and returned to baseline levels after 24 h. In addition, macrophage colony-stimulating factor (M-CSF) induced a dose-dependent induction of IP-10 mRNA in canine endothelial cells. M-CSF-mediated IP-10 expression peaked after 6 h of incubation and returned to baseline levels after 24 h. Canine endotoxemia is associated with a robust early expression of IP-10 in multiple tissues. IP-10 induction may be important in regulating lymphocyte recruitment and function. TNF-alpha, IL-1 beta, and M-CSF are potent inducers of IP-10 in canine endothelial cells and may indirectly mediate lymphocyte chemotaxis and activation in inflammatory processes.     

Does the frequency and avidity spectrum of the neuroantigen-specific T cells in the blood mirror the autoimmune process in the central nervous system of mice undergoing experimental allergic encephalomyelitis?

In humans, studies of autoreactive T cells that mediate multiple sclerosis have been largely confined to testing peripheral blood lymphocytes. Little is known how such measurements reflect the disease-mediating autoreactive T cells in the CNS. This information is also not available for murine experimental allergic encephalomyelitis (EAE); the low number of T cells that can be obtained from the blood or the brain of mice prevented such comparisons. We used single-cell resolution IFN-gamma ELISPOT assays to measure the frequencies and functional avidities of myelin basic protein (MBP:87-99)-specific CD4 cells in SJL mice immunized with this peptide. Functional MBP:87-99-specific IFN-gamma-producing cells were present in the CNS during clinical signs of EAE, but not during phases of recovery. In contrast, MBP:87-99-specific T cells persisted in the blood during all stages of the disease, and were also present in mice that did not develop EAE. Therefore, the increased frequency of MBP:87-99-reactive T cells in the blood reliably reflected the primed state, but not the inflammatory activity of these cells in the brain. The functional avidity of the MBP:87-99-reactive T cells was identical in the brain and blood and did not change over 2 mo as the mice progressed from acute to chronic EAE. Therefore, high-affinity T cells did not become selectively enriched in the target organ, and avidity maturation of the MBP:87-99-specific T cell repertoire did not occur in the observation period. The data may help the interpretation of measurements made with peripheral blood lymphocytes of multiple sclerosis patients.     

Selective modulation of MHC class II chaperons by a novel IFN-γ-inducible class II transactivator variant in lung adenocarcinoma A549 cells

Class II transactivator (CIITA) plays a critical role in controlling major histocompatibility complex (MHC) class II gene expression. In this study, two novel alternatively spliced variants of human interferon (IFN)-γ-inducible CIITA, one missing exon 7 (CIITAΔE7), the other with TAG inserted at exon 4/5 junction (CIITA-TAG), were identified and characterized. Both variants are naturally occurring since they are present in primary cells. Unlike CIITA-TAG, CIITAΔE7 is expressed more abundantly in lung adenocarcinoma A549 cells than in the non-transformed counterpart BEAS-2B cells following IFN-γ stimulation. Transfection experiments showed that CIITAΔE7 induced a markedly lower level of surface HLA-DR, -DP, -DQ expression than CIITA-TAG in A549 cells but not in BEAS-2B cells, although both variants elicited similar amounts of total DR, DP, and DQ proteins. This differential effect was correlated with, in A549 cells, decreased expression of Ii and HLA-DM genes, along with increased expression of HLA-DO genes. Ii and HLA-DM are chaperons assisting in HLA class II assembly, while HLA-DO functions to inhibit endosomal peptide loading and HLA class II membrane transport. These findings raise the possibility that CIITAΔE7 interacts with unknown cancer-associated factors to selectively modulate genes involved in the assembly and transport of HLA class II molecules.     

Improved survival in tumor-bearing SCID mice treated with interferon-γ-inducible protein 10 (IP-10/CXCL10)

Tumor growth requires angiogenesis, which in turn requires an imbalance in the presence of angiogenic and angiostatic factors. We have shown that the CXC chemokine family, consisting of members that are either angiogenic or angiostatic, is a major determinant of tumor-derived angiogenesis in non-small-cell lung cancer (NSCLC). Intratumor injection of interferon-inducible protein 10 (IP-10, or CXCL10), an angiostatic CXC chemokine, led to reduced tumor growth in a SCID mouse model of NSCLC. In this study, we hypothesized that treatment with CXCL10 would, by restoring the angiostatic balance, improve long-term survival in NSCLC-bearing SCID mice. To test this hypothesis, A549 NSCLC cells were injected in the subcutis of the flank, followed by intratumor injections with CXCL10 continuously (group I), or for ten weeks (group II), or a control group (human serum albumin). Median survival was 169, 130, and 86 days respectively (P<0.0001). We extended these studies to examine the mechanism of prolonged survival in CXCL10-treated mice. CXCL10 treatment inhibited lung metastases, but was dependent upon continued treatment, and was associated with an increased rate of apoptosis in the primary tumor, with no direct effect on the proliferation of the NSCLC cells. Furthermore, the inhibition of lung metastases was due to the angiostatic effect of CXCL10 on the primary tumor, since the rate of apoptosis within lung metastases was unaffected. These data suggest that anti-angiogenic therapy of human lung cancer should be continued indefinitely to realize persistent benefit, and confirms the anti-metastatic capacity of localized angiostatic therapy.     

ESAT6 differentially inhibits IFN-γ-inducible class II transactivator isoforms in both a TLR2-dependent and -independent manner

Mycobacterium tuberculosis (Mtb) downregulates the surface expression of major histocompatibility class II (MHC II) molecules on macrophages via modulating class II transactivator (CIITA) protein of the host cell. This results in decreased effector function of CD4(+) T cells. In macrophages, CIITA is transcribed by the promoters I (pI) and IV (pIV) and the corresponding gene products are referred to as type I and type IV CIITA, respectively. Earlier studies have mainly focused on CIITA transcribed by pIV; however, these studies also showed that type IV CIITA expression was transient and dispensable for MHC II expression. In the present study, we observed that the Mtb 6-kDa, early secreted antigen (ESAT6) inhibited interferon (IFN)-γ-induced type I as well as type IV CIITA, but, interestingly, inhibition of type I CIITA was found to be independent of Toll-like receptor-2 (TLR2), whereas that of type IV was TLR2 dependent. Moreover, we also present evidence to show that ESAT6-mediated inhibition was regulated via remodeling of the chromatin. We found that ESAT6 caused a decrease in the IFN-γ-stimulated methylation of the histone H3K4, as well as in the levels of histone acetylation at the CIITA pI locus in macrophages. We also found the involvement of mitogen-activated protein kinases ERK1/2 and p38 in the regulation of CIITA by ESAT6. In conclusion, our studies suggest that ESAT6 could inhibit the expression of type I and type IV CIITA through different pathways. Furthermore, ESAT6 could signal through putative receptors other than TLR2, and that the inhibition of IFN-γ-stimulated CIITA by ESAT6 was regulated at the chromatin level.     

Molecular cloning and expression analysis of the interferon-γ-inducible lysosomal thiol reductase gene from the shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

The interferon-γ-inducible lysosomal thiol reductase enzymes (GILT) have been shown to play an important role in the processing of exogenous antigens by catalyzing disulfide bond reduction, that facilitates unfolding of the native protein antigen to simplify further cleavage by cellular proteases. In this study a Penaeus monodon GILT (PmGILT) gene was isolated from an EST library of white spot syndrome virus (WSSV)-infected P. monodon. The full-length cDNA of the PmGILT gene was 780 bp and contained an open reading frame of 657 bp that encoded 218 amino acid residues with a predicted protein molecular weight of 24 kDa. The deduced amino acid sequence of PmGILT contains an active site CXXS motif, a GILT signature sequence (CQHGX₂ECX₂NX₄C) and 10 conserved cysteines together with other signature characteristics of GILT proteins. RT-PCR analysis showed that the PmGILT mRNA expression level was clearly up-regulated in the lymphoid organ of both the LPS-induced and WSSV-infected shrimp, compared to normal shrimp. In response to WSSV infection, the penaeid shrimp JAK/STAT pathway is reported to play an important role in the lymphoid organ. We hypothesize that this activated STAT may stimulate GILT expression so that it can be involved in the shrimp immune response system.     

The role of IFN-γ in regulation of IFN-γ-inducible protein 10 (IP-10) expression in lung epithelial cell and peripheral blood mononuclear cell co-cultures

Background

Epithelial to mesenchymal transition (EMT) in alveolar epithelial cells (AECs) has been widely observed in patients suffering interstitial pulmonary fibrosis. In vitro studies have also demonstrated that AECs could convert into myofibroblasts following exposure to TGF-β1. In this study, we examined whether EMT occurs in bleomycin (BLM) induced pulmonary fibrosis, and the involvement of bronchial epithelial cells (BECs) in the EMT. Using an α-smooth muscle actin-Cre transgenic mouse (α-SMA-Cre/R26R) strain, we labelled myofibroblasts in vivo. We also performed a phenotypic analysis of human BEC lines during TGF-β1 stimulation in vitro.

Methods

We generated the α-SMA-Cre mouse strain by pronuclear microinjection with a Cre recombinase cDNA driven by the mouse α-smooth muscle actin (α-SMA) promoter. α-SMA-Cre mice were crossed with the Cre-dependent LacZ expressing strain R26R to produce the double transgenic strain α-SMA-Cre/R26R. β-galactosidase (βgal) staining, α-SMA and smooth muscle myosin heavy chains immunostaining were carried out simultaneously to confirm the specificity of expression of the transgenic reporter within smooth muscle cells (SMCs) under physiological conditions. BLM-induced peribronchial fibrosis in α-SMA-Cre/R26R mice was examined by pulmonary βgal staining and α-SMA immunofluorescence staining. To confirm in vivo observations of BECs undergoing EMT, we stimulated human BEC line 16HBE with TGF-β1 and examined the localization of the myofibroblast markers α-SMA and F-actin, and the epithelial marker E-cadherin by immunofluorescence.

Results

βgal staining in organs of healthy α-SMA-Cre/R26R mice corresponded with the distribution of SMCs, as confirmed by α-SMA and SM-MHC immunostaining. BLM-treated mice showed significantly enhanced βgal staining in subepithelial areas in bronchi, terminal bronchioles and walls of pulmonary vessels. Some AECs in certain peribronchial areas or even a small subset of BECs were also positively stained, as confirmed by α-SMA immunostaining. In vitro, addition of TGF-β1 to 16HBE cells could also stimulate the expression of α-SMA and F-actin, while E-cadherin was decreased, consistent with an EMT.

Conclusion

We observed airway EMT in BLM-induced peribronchial fibrosis mice. BECs, like AECs, have the capacity to undergo EMT and to contribute to mesenchymal expansion in pulmonary fibrosis.     

Identification of novel mimicry epitopes for cardiac myosin heavy chain-α that induce autoimmune myocarditis in A/J mice

Myocarditis is one cause of sudden cardiac death in young adolescents, and individuals affected with myocarditis can develop dilated cardiomyopathy, a frequent reason for heart transplantation. Exposure to environmental microbes has been suspected in the initiation of heart autoimmunity, but the direct causal link is lacking. We report here identification of novel mimicry epitopes that bear sequences similar to those in cardiac myosin heavy chain (MYHC)-α 334–352. These epitopes represent Bacillus spp., Magnetospirillum gryphiswaldense, Cryptococcus neoformans and Zea mays. The mimicry peptides induced varying degrees of myocarditis in A/J mice reminiscent of the disease induced with MYHC-α 334–352. We demonstrate that the mimics induce cross-reactive T cell responses for MYHC-α 334–352 as verified by MHC class II IA^k/tetramer staining and Th-1 and Th-17 cytokines similar to those of MYHC-α 334–352. The data suggest that exposure to environmental microbes which are otherwise innocuous can predispose to heart autoimmunity by molecular mimicry.     

MMP-12-mediated by SARM-TRIF signaling pathway contributes to IFN-γ-independent airway inflammation and AHR post RSV infection in nude mice

Background

Respiratory syncytial virus (RSV) is one of the most frequently observed pathogens during infancy and childhood. However, the corresponding pathogenesis has not been determined to date. We previously demonstrated that IFN-γ plays an important role in RSV pathogenesis, and SARM-TRIF-signaling pathway could regulate the production of IFN-γ. This study is to investigate whether T cells or innate immune cells are the predominant producers of IFN-γ, and further to explore other culprits in addition to IFN-γ in the condition of RSV infection.

Methods

Normal BALB/c mice and nude mice deficient in T cells were infected intranasally with RSV. Leukocytes in bronchoalveolar lavage fluid were counted, lung histopathology was examined, and airway hyperresponsiveness (AHR) was measured by whole-body plethysmography. IFN-γ and MMP-12 were detected by ELISA. MMP408, a selective MMP-12 inhibitor, was given intragastrically. Resveratrol, IFN-γ neutralizing antibody and recombinant murine IFN-γ were administered intraperitoneally. SARM and TRIF protein were semi-quantified by Western blot. siRNA was used to knock-down SARM expression.

Results

RSV induced significant airway inflammation and AHR in both mice; IFN-γ was significantly increased in BALB/c mice but not in nude mice. MMP-12 was dramatically increased in both mice but earlier in nude mice. When MMP-12 was inhibited by MMP408, RSV-induced respiratory symptoms were alleviated. SARM was significantly suppressed while TRIF was significantly enhanced in both mice strains. Following resveratrol administration in nude mice, 1) SARM inhibition was prevented, 2) TRIF and MMP-12 were correspondingly down-regulated and 3) airway disorders were subsequently alleviated. Moreover, when SARM was efficiently knocked down using siRNA, TRIF and MMP-12 were markedly enhanced, and the anti-RSV effects of resveratrol were remarkably abrogated. MMP-12 was significantly increased in the IFN-γ neutralizing antibody-treated BALB/c mice but reduced in the recombinant murine IFN-γ-treated nude mice.

Conclusions

MMP-12 can result in at least part of the airway inflammation and AHR independent of IFN-γ. And SARM-TRIF- signaling pathway is involved in regulating the overproduction of MMP-12. To the best of our knowledge, this study is the first that has examined the effects of SARM on MMP-12 and further highlights the potential to target SARM-TRIF-MMP-12 cascades to treat RSV infection.

Electronic supplementary material

The online version of this article (doi:10.1186/s12931-015-0176-8) contains supplementary material, which is available to authorized users.     

Metallic gold beads in hyaluronic acid: a novel form of gold-based immunosuppression? Investigations of the immunosuppressive effects of metallic gold on cultured J774 macrophages and on neuronal gene expression in experimental autoimmune encephalomyelitis

Multiple sclerosis (MS) is a neurodegenerative disease caused by recurring attacks of neuroinflammation leading to neuronal death. Immune-suppressing gold salts are used for treating connective tissue diseases; however, side effects occur from systemic spread of gold ions. This is limited by exploiting macrophage-induced liberation of gold ions (dissolucytosis) from gold surfaces. Injecting gold beads in hyaluronic acid (HA) as a vehicle into the cavities of the brain can delay clinical signs of disease progression in the MS model, experimental autoimmune encephalitis (EAE). This study investigates the anti-inflammatory properties of metallic gold/HA on the gene expression of tumor necrosis factor (Tnf-α), Interleukin (Il)-1β, Il-6, Il-10, Colony-stimulating factor (Csf)-v2, Metallothionein (Mt)-1/2, Bcl-2 associated X protein (Bax) and B cell lymphoma (Bcl)-2 in cultured J774 macrophages and in rodents with early stages of EAE. Cells grew for 5 days on gold/HA or HA, then receiving 1,000 ng/mL lipopolysaccharide (LPS) as inflammatory challenge. In the EAE experiment, 12 Lewis rats received gold injections and control groups included 11 untreated and 12 HA-treated EAE rats and five healthy animals. The experiment terminated day 9 when the first ten animals showed signs of EAE, only one of which were gold-treated (1p = 0.0367). Gene expression in the macrophages showed a statistically significant decrease in Il-6, Il-1β and Il-10-response to LPS; interestingly HA induced a statistically significant increase of Il-10. In the EAE model gene expression of inflammatory cytokines increased markedly. Compared to EAE controls levels of Tnf-α, Il-1β, Il-10, Il-6, IL-2, Ifn-γ, Il-17, transforming growth factor (Tgf)-β, superoxide dismutase (Sod)-2, Mt-2 and fibroblast growth factor (Fgf)-2 were lower in the gold-treated group. HA-treated animals expressed similar or intermediate levels. Omnibus testing for reduced inflammatory response following gold-treatment was not significant, but tendencies towards a decrease in the Sod-2, Fgf-2, Il-1β response and a higher Bdnf and IL-23 gene expression were seen. In conclusion, our findings support that bio-liberation of gold from metallic gold surfaces have anti-inflammatory properties similar to classic gold compounds, warranting further studies into the pharmacological potential of this novel gold-treatment and the possible synergistic effects of hyaluronic acid.