Attenuated accumulation of regulatory T cells and reduced production of interleukin 10 lead to the exacerbation of tissue injury in a mouse model of acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is a pathological condition that involves diffuse lung injury and severe hypoxemia caused by pulmonary and systemic diseases. We have established a mouse model of severe ARDS, developed by intratracheal injection of α‐galactosylceramide (α‐GalCer), an activator of natural killer T (NKT) cells, followed by LPS. In the present study, we used this model to investigate the regulatory mechanism in the early inflammatory response during acute lung injury. In α‐GalCer/LPS‐treated mice, the number of CD4⁺CD25⁺Foxp3⁺ regulatory T (Treg) cells and the expression of a Treg cell‐tropic chemokine, secondary lymphoid‐tissue chemokine (SLC), in the lungs was significantly lower than in mice treated with LPS alone. Giving recombinant (r)SLC increased the number of Treg cells in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced the expression of SLC and the accumulation of Treg cells in the lungs of α‐GalCer/LPS‐treated mice, whereas giving recombinant (r)IFN‐γ reduced the number of Treg cells in mice treated with LPS alone. IL‐10 production was significantly lower in α‐GalCer/LPS‐treated mice than in mice treated with LPS alone. Giving rIL‐10 prolonged survival and attenuated lung injury as a result of reduced production of inflammatory cytokines (such as IL‐1β, IL‐6, TNF‐α, and IFN‐γ) and chemokines (including MCP‐1, RANTES, IP‐10, Mig, MIP‐2, and KC) in α‐GalCer/LPS‐treated mice. Treatment with anti‐IFN‐γ mAb enhanced IL‐10 production in α‐GalCer/LPS‐treated mice. These results suggest that the attenuated accumulation of Treg cells may be involved in the development of severe ARDS through a reduction in the synthesis of IL‐10.

     

IL‐27 Rα+ cells promoted allorejection via enhancing STAT1/3/5 phosphorylation

Recently, emerging evidence strongly suggested that the activation of interleukin‐27 Receptor α (IL‐27Rα) could modulate different inflammatory diseases. However, whether IL‐27Rα affects allotransplantation rejection is not fully understood. Here, we investigated the role of IL‐27Rα on allorejection both in vivo and in vitro. The skin allotransplantation mice models were established, and the dynamic IL‐27Rα/IL‐27 expression was detected, and IL‐27Rα⁺ spleen cells adoptive transfer was performed. STAT1/3/5 phosphorylation, proliferation and apoptosis were investigated in mixed lymphocyte reaction (MLR) with recombinant IL‐27 (rIL‐27) stimulation. Finally, IFN‐γ/ IL‐10 in graft/serum from model mice was detected. Results showed higher IL‐27Rα/IL‐27 expression in allografted group compared that syngrafted group on day 10 (top point of allorejection). IL‐27Rα⁺ spleen cells accelerated allograft rejection in vivo. rIL‐27 significantly promoted proliferation, inhibited apoptosis and increased STAT1/3/5 phosphorylation of alloreactive splenocytes, and these effects of rIL‐27 could be almost totally blocked by JAK/ STAT inhibitor and anti‐IL‐27 p28 Ab. Finally, higher IL‐27Rα⁺IFN‐γ⁺ cells and lower IL‐27Rα⁺IL‐10⁺ cells within allografts, and high IFN‐γ/low IL‐10 in serum of allorejecting mice were detected. In conclusion, these data suggested that IL‐27Rα⁺ cells apparently promoted allograft rejection through enhancing alloreactive proliferation, inhibiting apoptosis and up‐regulating IFN‐γ via enhancing STAT pathway. Blocking IL‐27 pathway may favour to prevent allorejection, and IL‐27Rα may be as a high selective molecule for targeting diagnosis and therapy for allotransplantation rejection.     

The immune characterization of interferon‐β responses in tuberculosis patients

We aimed to assess the immunoregulatory effects of IFN‐β in patients with tuberculous pleurisy. IFN‐β, IFN‐γ and IL‐17 expression levels were detected, and correlations among these factors in different culture groups were analyzed. Pleural fluid mononuclear cells (PFMC) from tuberculous pleural effusions, but not peripheral blood mononuclear cells (PBMC) from healthy donors, spontaneously expressed IFN‐β, IL‐17 and IFN‐γ. Moreover, exogenous IFN‐β significantly inhibited the expression of IL‐17 in PFMC. By contrast, IFN‐β simultaneously enhanced the levels of IFN‐γ. To further investigate the regulation of IL‐17 and IFN‐γ by endogenous IFN‐β, an IFN‐β neutralizing antibody was simultaneously added to bacillus Calmette‐Guérin (BCG)‐stimulated PFMC. IL‐17 expression was significantly increased, but IFN‐γ production was markedly decreased in the experimental group supplemented with the IFN‐β neutralizing antibody. Simultaneously, IL‐17 production was remarkably increased in the experimental group supplemented with the IFN‐γ neutralizing antibody. Taken together, in our study, we first found that freshly isolated PFMC, but not PBMC from healthy donors, spontaneously expressed IFN‐β, IL‐17 and IFN‐γ in vivo. Moreover, IFN‐β suppressed IL‐17 expression and increased IFN‐γ production. Furthermore, both IFN‐β and IFN‐γ down‐regulated IL‐17 expression. These observations suggest that caution is required when basing anti‐tuberculosis treatment on the inhibition of IFN‐β signaling.     

Consecutive oral administration of Bifidobacterium longum MM‐2 improves the defense system against influenza virus infection by enhancing natural killer cell activity in a murine model

Bifidobacterium, one of the major components of intestinal microflora, shows anti‐influenza virus (IFV) potential as a probiotic, partly through enhancement of innate immunity by modulation of the intestinal immune system. Bifidobacterium longum MM‐2 (MM‐2), a very safe bacterium in humans, was isolated from healthy humans and its protective effect against IFV infection in a murine model shown. In mice that were intranasally inoculated with IFV, oral administration of MM‐2 for 17 consecutive days improved clinical symptoms, reduced mortality, suppressed inflammation in the lower respiratory tract, and decreased virus titers, cell death, and pro‐inflammatory cytokines such as IL‐6 and TNF‐α in bronchoalveolar lavage fluid. The anti‐IFV mechanism of MM‐2 involves innate immunity through significant increases in NK cell activities in the lungs and spleen and a significant increase in pulmonary gene expression of NK cell activators such as IFN‐γ, IL‐2, IL‐12 and IL‐18. Even in non‐infected mice, MM‐2 administration also induced significant enhancement of both IFN‐γ production by Peyer's patch cells (PPs) and splenetic NK cell activity. Oral administration of MM‐2 for 17 days activates systemic immunoreactivity in PPs, which contributes to innate immunity, including NK cell activation, resulting in an anti‐IFV effect. MM‐2 as a probiotic may function as a prophylactic agent in the management of an IFV epidemic.     

Expression of bovine (Bos indicus) interleukin‐18 in Escherichia coli and its biological activity

IL‐18 modulates immune functions by inducing IFN‐γ production and promoting Th1 immune responses. In the present study, we amplified and cloned the sequence (582 bp) encoding full‐length bovine IL‐18 from PBMC stimulated with PHA. The nucleotide and the deduced amino acid sequence of Bos indicus IL‐18 showed an identity of 86–98% compared with IL‐18 sequences of other ruminants. The insert was subcloned into a pET 32a vector and expressed in Escherichia coli as a fusion protein and the matured protein was obtained by caspase I treatment. The specificity of these proteins was confirmed by western blotting. The biological activity of the purified protein was analyzed by its ability to induce IFN‐γ production in PBMC measured by ELISA and qPCR.     

Anti‐arthritis activity of cationic materials

Cationic materials exhibit remarkable anti‐inflammatory activity in experimental arthritis models. Our aim was to confirm this character of cationic materials and investigate its possible mechanism. Adjuvant‐induced arthritis (AIA) models were used to test cationic materials for their anti‐inflammatory activity. Cationic dextran (C‐dextran) with different cationic degrees was used to investigate the influence of the cationic elements of materials on their anti‐inflammatory ability. Peritoneal macrophages and spleen cells were used to test the expression of cytokines stimulated by cationic materials. Interferon (IFN)‐γ receptor‐deficient mice and macrophage‐depleted rats were used to examine the possible mechanisms of the anti‐inflammatory activity of cationic materials. In AIA models, different cationic materials shared similar anti‐inflammatory characters. The anti‐inflammatory activity of C‐dextran increased with as the cationic degree increased. Cationic materials stimulated interleukin (IL)‐12 expression in peritoneal macrophages, and strong stimulation of IFN‐γ secretion was subsequently observed in spleen cells. In vivo experiments revealed that circulating IL‐12 and IFN‐γ were enhanced by the cationic materials. Using IFN‐γ receptor knockout mice and macrophage‐depleted rats, we found that IFN‐γ and macrophages played key roles in the anti‐inflammatory activity of the materials towards cells. We also found that neutrophil infiltration at inflammatory sites was reduced when AIA animals were treated with C‐dextran. We propose that cationic signals act through an unknown receptor on macrophages to induce IL‐12 secretion, and that IL‐12 promotes the expression of IFN‐γ by natural killer cells (or T cells). The resulting elevated systemic levels of IFN‐γ inhibit arthritis development by preventing neutrophil recruitment to inflammatory sites.     

Helicobacter pylori modulation of gastric and duodenal mucosal T cell cytokine secretions in children compared with adults

Background. In contrast to adults, ulcers are un‐common in Helicobacter pylori‐infected children. Since immunological determinants influence the outcome of H. pylori infection, we have investigated mucosal T cell responses in H. pylori‐infected children and compared them with those of adults and negative controls. Material and Methods. Mucosal biopsies were obtained from 43 patients undergoing an upper GI endoscopy for dyspeptic symptoms. The concentrations of released cytokines and the density of CD3⁺, CD25⁺ and CD69⁺cells were evaluated by flow cytometry, and the numbers of cytokine‐secreting cells were measured by ELISPOT. Results. The numbers of isolated antral CD3⁺ lymphocytes were only significantly raised in infected adults compared with noninfected controls (p < 0.05), whereas the proportion of CD3⁺ cells expressing activation markers (CD25 or CD69) remained low. In the stomach, IFN‐γ concentrations increased in infected children and infected adults compared with controls (p < 0.05), but IFN‐γ concentrations were tenfold lower in children than in adults (p < 0.01). IL‐2, IL‐4, IL‐10 and TNF‐α concentrations were similar in infected and in uninfected children and adults. In contrast, in the duodenum, IFN‐γ, as well as IL‐4 and IL‐10 concentrations were only increased in infected children compared with controls (p < 0.05). The concentrations of these cytokines were similar in both groups of adults who, however, like children, displayed a higher number of duodenal IL‐4‐secreting cells compared to controls (p < 0.05). Conclusion. These results suggest that IFN‐γ secretion in the stomach of H. pylori‐infected patients is lower in children than in adults. This could protect children from development of severe gastro‐duodenal diseases such as ulcer disease. In addition, infected patients are characterised by a dysregulation of the mucosal cytokine secretion at distance from the infection site.     

Evaluation of a New Tumor Necrosis Factor-α-Inducing Membrane Protein of Helicobacter pylori as a Prophylactic Vaccine Antigen

Background: Tumor necrosis factor (TNF)‐α‐inducing protein (Tipα) is a newly identified carcinogenic factor present in Helicobacter pylori. Tipα has the unique function of inducing TNF‐α production by gastric cells in vitro and is assumed to be related with the development of gastritis and gastric cancer. We investigated the effects of vaccination with Tipα against H. pylori infection and analyzed the immune responses. Methods: C57BL/6 mice were immunized via the intranasal route with CpG, recombinant Tipα + CpG, and recombinant del‐Tipα (a mutant of Tipα) + CpG. Eight weeks after the mice were infected with H. pylori (5 × 10⁷ CFU), the number of colonizing bacteria in the stomach was calculated, and the histological severity of gastritis was evaluated. Levels of Tipα‐specific IgG and IgA antibodies in mouse serum were measured by an enzyme‐linked immunosorbent assay (ELISA). Local production of cytokines including Interleukin (IL)‐10, TNF‐α and Interferon (IFN)‐γ in gastric mucosa was also measured by real time‐PCR. Results: Levels of Tipα‐specific antibodies were significantly higher in Tipα‐immunized and del‐Tipα‐immunized mice than in the infection control group. The numbers of colonizing bacteria were significantly reduced in Tipα‐immunized mice (4.29 × 10⁵ CFU/g) and del‐Tipα immunized mice (2.5 × 10⁵ CFU/g) compared with infection control mice (5.7 × 10⁶ CFU/g). The levels of IFN‐γ and IL‐10 were significantly higher in del‐Tipα‐immunized mice than the infection control group. Conclusion: Vaccinations with Tipα and del‐Tipα were effective against H. pylori infection. The inhibition of H. pylori colonization is associated mainly with Th1 cell‐mediated immunity.     

IL‐12 stimulates the osteoclast inhibitory peptide‐1 (OIP‐1/hSca) gene expression in CD4+ T cells

Immune cell products such as interferon (IFN)‐γ and interleukin (IL)‐12 are potent inhibitors of osteoclast formation. We previously characterized the human osteoclast inhibitory peptide‐1 (OIP‐1/hSca), a Ly‐6 gene family member and showed IFN‐γ modulation of OIP‐1 expression in bone marrow cells. Whether, IL‐12 regulates OIP‐1 expression in the bone microenvironment is unclear. Real‐time PCR analysis revealed that IL‐12 treatment significantly enhanced OIP‐1 mRNA expression in human bone marrow mononuclear cells. Because IL‐12 induces IFN‐γ production by T cells, we tested whether IFN‐γ participates in IL‐12 stimulation of OIP‐1 gene expression in these cells. IL‐12 treatment in the presence of IFN‐γ neutralizing antibody significantly increased OIP‐1 mRNA expression, suggesting that IL‐12 directly regulates OIP‐1 gene expression. Interestingly, real‐time PCR analysis demonstrated that IL‐12 induces OIP‐1 expression (3.2‐fold) in CD4+ T cells; however, there was no significant change in CD8+ T cells. Also, IL‐12 (10 ng/ml) treatment of Jurkat cells transfected with OIP‐1 gene (?1 to ?1,988 bp) promoter‐luciferase reporter plasmid demonstrated a 5‐fold and 2.7‐fold increase in OIP‐1 gene promoter activity in the presence and absence of antibody against IFN‐γ, respectively. We showed that STAT‐1,3 inhibitors treatment significantly decreased IL‐12 stimulated OIP‐1 promoter activity. Chromatin immunoprecipitation (ChIP) assay confirmed STAT‐3, but not STAT‐1 binding to the OIP‐1 gene promoter in response to IL‐12 stimulation. These results suggest that IL‐12 stimulates the OIP‐1 gene expression through STAT‐3 activation in CD4+ T cells. J. Cell. Biochem. 107: 104–111, 2009. © 2009 Wiley‐Liss, Inc.     

Modulatory effects of interferon‐γ and interleukin‐4 on cellular immune responses against Hypoderma lineatum antigens

This study investigates the in vitro modulatory effects of interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) on both proliferative bovine T cell responses and IL‐10 production induced by different antigens [crude larval extract and the purified fractions hypodermin A, B and C (HyA, HyB, HyC)] obtained from first instars of Hypoderma lineatum (Diptera: Oestridae), alone or in the presence of the mitogen concanavalin A. Incubation with the different parasitic antigens resulted in significant inhibition of T cell proliferation and IL‐10 production, which, in general, did not revert after the addition of IFN‐γ and IL‐4. In the absence of antigens, IL‐4 induced significant inhibition of mitogen‐induced T cell responses. Exogenous IFN‐γ exhibited an inhibitory effect on cell proliferation in the presence of the purified fractions HyB and HyC. These in vitro data suggest that far from neutralizing the effects of larval antigens, the addition of IFN‐γ potentiates their anti‐proliferative activity; by contrast, IL‐4 had no consistent effects on proliferative responses to Hypoderma. IL‐4 provoked an increment of IL‐10 levels in supernatants of HyB‐stimulated cells. In conclusion, exogenous IFN‐γ and IL‐4 were unable to counteract the suppressor effects of H. lineatum antigens.     

Influence of orally administered Lactobacillus GG on respiratory immune response in a murine model of diet‐induced obesity

Mice with diet‐induced obesity were fed with Lactobacillus rhamnosus GG (LGG) suspended in saline or saline alone (control mice). Pulmonary mRNA expression of IFN‐γ; IFN‐α receptor 1; CD247 antigen; killer cell lectin‐like receptor subfamily K, member 1; TNF‐α; IL‐12 receptor β1 and IL‐2 receptor β, and the proportion of Lactobacillales in feces were significantly greater in the LGG group than in the control mice (P < 0.05 and P < 0.01, respectively). These results suggest that LGG alters the respiratory immunity of obese subjects through having a potent impact on intestinal immunity.     

Discordant susceptibility of inbred C57BL/6 versus outbred CD1 mice to experimental fungal sepsis

Individual susceptibility differences to fungal infection following invasive and/or immunosuppressive medical interventions are an important clinical issue. In order to explore immune response‐related factors that may be linked to fungal infection susceptibility, we have compared the response of inbred C57BL/6J and outbred CD1 mouse strains to different experimental models of fungal sepsis. The challenge of animals with the zymosan‐induced generalised inflammation model revealed poorer survival rates in C57BL/6J, consistent with lower Th1 cytokine interferon (IFN)‐γ serum levels, compared with CD1 mice. Likewise, ex vivo exposure of C57BL/6J splenocytes to zymosan but also bacterial lipopolisaccharide or lipoteichoic acid, resulted in lower IFN‐γ secretion compared with CD1 mice. C57BL/6J susceptibility could be reverted by rescue infusion of relative low IFN‐γ doses (0.2 μg/kg) either alone or in combination with the ß‐glucan‐binding CD5 protein (0.7 mg/kg) leading to improved post zymosan‐induced generalised inflammation survival. Similarly, low survival rates to systemic Candida albicans infection (2.86 × 10⁴ CFU/gr) were ameliorated by low‐dose IFN‐γ infusion in C57BL/6J but not CD1 mice. Our results highlight the importance of strain choice in experimental fungal infection models and provide a susceptibility rationale for more specific antifungal immunotherapy designs.     

TLR9 limits enteric antimicrobial responses and promotes microbiota‐based colonisation resistance during Citrobacter rodentium infection

Mammalian cells express an array of toll‐like receptors to detect and respond to microbial pathogens, including enteropathogenic and enterohemorrhagic Escherichia coli (EPEC and EHEC). These clinically important attaching and effacing (A/E) pathogens infect the apical surface of intestinal epithelial cells, causing inflammation as well as severe diarrheal disease. Because EPEC and EHEC are human‐specific, the related murine pathogen Citrobacter rodentium has been widely used to define how hosts defend against A/E pathogens. This study explored the role of TLR9, a receptor that recognises unmethylated CpG dinucleotides present in bacterial DNA, in promoting host defence against C. rodentium. Infected Tlr9^?/? mice suffered exaggerated intestinal damage and carried significantly higher (10–100 fold) pathogen burdens in their intestinal tissues as compared with wild type (WT) mice. C. rodentium infection also induced increased antimicrobial responses, as well as hyperactivation of NF‐κB signalling in the intestines of Tlr9^?/? mice. These changes were associated with accelerated depletion of the intestinal microbiota in Tlr9^?/? mice as compared with WT mice. Notably, antibiotic‐based depletion of the gut microbiota in WT mice prior to infection increased their susceptibility to the levels seen in Tlr9^?/? mice. Our results therefore indicate that TLR9 signalling suppresses intestinal antimicrobial responses, thereby promoting microbiota‐mediated colonisation resistance against C. rodentium infection.     

NOD1 is required for Helicobacter pylori induction of IL‐33 responses in gastric epithelial cells

Helicobacter pylori (H. pylori) causes chronic inflammation which is a key precursor to gastric carcinogenesis. It has been suggested that H. pylori may limit this immunopathology by inducing the production of interleukin 33 (IL‐33) in gastric epithelial cells, thus promoting T helper 2 immune responses. The molecular mechanism underlying IL‐33 production in response to H. pylori infection, however, remains unknown. In this study, we demonstrate that H. pylori activates signalling via the pathogen recognition molecule Nucleotide‐Binding Oligomerisation Domain‐Containing Protein 1 (NOD1) and its adaptor protein receptor‐interacting serine–threonine Kinase 2, to promote production of both full‐length and processed IL‐33 in gastric epithelial cells. Furthermore, IL‐33 responses were dependent on the actions of the H. pylori Type IV secretion system, required for activation of the NOD1 pathway, as well as on the Type IV secretion system effector protein, CagA. Importantly, Nod1^+/+ mice with chronic H. pylori infection exhibited significantly increased gastric IL‐33 and splenic IL‐13 responses, but decreased IFN‐γ responses, when compared with Nod1^?/? animals. Collectively, our data identify NOD1 as an important regulator of mucosal IL‐33 responses in H. pylori infection. We suggest that NOD1 may play a role in protection against excessive inflammation.     

Modulation of the inflammation–coagulation interaction during pneumococcal pneumonia by immunobiotic Lactobacillus rhamnosus CRL1505: Role of Toll‐like receptor 2

The present study evaluated the effect of nasally given Lactobacillus rhamnosus CRL1505 on the immunocoagulative response during pneumococcal infection in immunocompetent mice. In addition, we aimed to gain insight into the mechanism involved in the immunomodulatory effect of the L. rhamnosus CRL1505 strain by evaluating the role of TLR2. Results showed that nasally given L. rhamnosus CRL1505 effectively regulates inflammation and hemostatic alterations during the pneumococcal infection. Immunobiotic treatment significantly reduced permeability of the bronchoalveolar–capillary barrier, and general cytotoxicity, decreasing lung tissue damage. The CRL1505 strain improved the production of TNF‐α, IFN‐γ, and IL‐10 after pneumococcal challenge. In addition, increased TM and TF expressions were found in lungs of L. rhamnosus CRL1505‐treated mice. Moreover, we demonstrated, for the first time, that the TLR2 signaling pathway has a role in the induction of IFN‐γ and IL‐10 and in the reduction of TF. The results also allow us to speculate that a PRR, other than TLR2, may mediate the immunobiotic activity of L. rhamnosus CRL1505 and could explain changes in TNF‐α and TM.     

Prion protein participates in the regulation of classical and alternative activation of BV2 microglia

The cellular prion protein (PrP^C) is a glycoprotein anchored by glycosylphosphatidylinositol (GPI) to the cell surface and is abundantly expressed in the central nervous system. Numerous studies have suggested a protective function for PrP^C, including protection from ischemic and excitotoxic lesions and several apoptotic insults, and recent reports have shown that PrP^C has a context‐dependent neuroprotective function. In this study, we investigated the effect of PPNP down‐regulation on various forms of microglial activation. We first examined the mRNA expression of PRNP upon exposure to IFN‐γ, IL‐4, or IL‐10 in BV2 microglia. We then analyzed the effect of si‐RNA‐mediated disruption of PRNP on different parameters of microglial activation in IFN‐γ‐, IL‐4‐, or IL‐10‐stimulated microglia. The results showed that PRNP mRNA expression was invariably down‐regulated in microglia upon exposure to IFN‐γ, IL‐4, or IL‐10. PRNP silencing prior to cytokines treatment reduced the responsiveness of microglia to INF‐γ treatment, significantly altered IL‐4‐induced microglial activation phenotype, and had no effect on IL‐10‐induced microglial activation. Together, these results support a role of PrP^C in the modulation of the shift of microglia from a quiescent state to an activated phenotype and in the regulation of the microglial response during classical and alternative activation.