白细胞介素-6在急性胰腺炎中的作用及其机制研究

目的:明确白细胞介素-6(IL-6)在小鼠急性胰腺炎中的作用及其机制研究。方法:通过胰胆管结扎的方法诱导小鼠急性胰腺炎;分离小鼠胰腺腺泡细胞。采用ELISA方法检测胰腺组织或腺泡细胞裂解物中的细胞因子;通过western blot分析检测组织或细胞中IL-6或ERK表达。结果:IL-6浓度在胰腺组织和腺泡细胞中显著增加(P0.05)。在离体原代小鼠腺泡细胞,TNF-α刺激增加IL-6释放(P0.05);与此同时,IL-6刺激可增加其它促炎性细胞因子的释放,两者都涉及ERK MAP激酶通路。黄酮类化合物木犀草素抑制IL-6刺激引起白细胞介素-6(IL-6)和人巨嗜细胞激活蛋白-1(CCL2/MCP-1)释放。最后进一步证实,IL-6激活人胰腺组织中的ERK。结论:IL-6在急性胰腺炎中增加,激活炎症通路并加重急性胰腺炎。     

IL-26 Is Overexpressed in Rheumatoid Arthritis and Induces Proinflammatory Cytokine Production and Th17 Cell Generation

Interleukin-26 (IL-26), a member of the IL-10 cytokine family, induces the production of proinflammatory cytokines by epithelial cells. IL-26 has been also reported overexpressed in Crohn''s disease, suggesting that it may be involved in the physiopathology of chronic inflammatory disorders. Here, we have analyzed the expression and role of IL-26 in rheumatoid arthritis (RA), a chronic inflammatory disorder characterized by joint synovial inflammation. We report that the concentrations of IL-26 are higher in the serums of RA patients than of healthy subjects and dramatically elevated in RA synovial fluids compared to RA serums. Immunohistochemistry reveals that synoviolin⁺ fibroblast-like synoviocytes and CD68⁺ macrophage-like synoviocytes are the main IL-26-producing cells in RA joints. Fibroblast-like synoviocytes from RA patients constitutively produce IL-26 and this production is upregulated by IL-1-beta and IL-17A. We have therefore investigated the role of IL-26 in the inflammatory process. Results show that IL-26 induces the production of the proinflammatory cytokines IL-1-beta, IL-6, and tumor necrosis factor (TNF)-alpha by human monocytes and also upregulates the expression of numerous chemokines (mainly CCL20). Interestingly, IL-26-stimulated monocytes selectively promote the generation of RORgamma t⁺ Th17 cells, through IL-1-beta secretion by monocytes. More precisely, IL-26-stimulated monocytes switch non-Th17 committed (IL-23R⁻ or CCR6⁻ CD161⁻) CD4⁺ memory T cells into Th17 cells. Finally, synovial fluids from RA patients also induce Th17 cell generation and this effect is reduced after IL-26 depletion. These findings show that IL-26 is constitutively produced by RA synoviocytes, induces proinflammatory cytokine secretion by myeloid cells, and favors Th17 cell generation. IL-26 thereby appears as a novel proinflammatory cytokine, located upstream of the proinflammatory cascade, that may constitute a promising target to treat RA and chronic inflammatory disorders.     

Coxsackievirus Infection Induces Autophagy-Like Vesicles and Megaphagosomes in Pancreatic Acinar Cells In Vivo

Autophagy can play an important part in protecting host cells during virus infection, and several viruses have developed strategies by which to evade or even exploit this homeostatic pathway. Tissue culture studies have shown that poliovirus, an enterovirus, modulates autophagy. Herein, we report on in vivo studies that evaluate the effects on autophagy of coxsackievirus B3 (CVB3). We show that in pancreatic acinar cells, CVB3 induces the formation of abundant small autophagy-like vesicles and permits amphisome formation. However, the virus markedly, albeit incompletely, limits the fusion of autophagosomes (and/or amphisomes) with lysosomes, and, perhaps as a result, very large autophagy-related structures are formed within infected cells; we term these structures megaphagosomes. Ultrastructural analyses confirmed that double-membraned autophagy-like vesicles were present in infected pancreatic tissue and that the megaphagosomes were related to the autophagy pathway; they also revealed a highly organized lattice, the individual components of which are of a size consistent with CVB RNA polymerase; we suggest that this may represent a coxsackievirus replication complex. Thus, these in vivo studies demonstrate that CVB3 infection dramatically modifies autophagy in infected pancreatic acinar cells.Macroautophagy—henceforth referred to as autophagy—is an intracellular process that is important for cellular differentiation, homeostasis, and survival. Through autophagy, long-lived cytosolic proteins and organelles become encapsulated within double-membraned vesicles, called autophagosomes, which fuse with lysosomes to facilitate degradation of protein and cellular organelles and to promote nutrient recycling/regeneration. Autophagy plays a key role in the host immune response to infection by viruses, bacteria, fungi, and parasites (reviewed in references 10 and 62). Within virus-infected cells, whole virions and/or viral proteins and nucleic acids are captured inside autophagosomes and degraded (following lysosomal fusion) through the process of xenophagy. Moreover, autophagosome fusion with the endosomal/lysosomal pathway facilitates Toll-like receptor recognition of viral materials and delivers endogenous cytosolic viral proteins to the major histocompatibility complex (MHC) class II antigen presentation pathway, which in turn may help to trigger activation of innate immunity (and type I interferon production) and promote antigen presentation to virus-specific CD4⁺ T cells (reviewed in references 9, 41, 44, 47, 72, and 90). A recent study has shown that autophagy is also involved in the processing and presentation of MHC class I-restricted viral epitopes (13).Given the importance of autophagy in antiviral immunity, it is perhaps not surprising that viruses have evolved mechanisms to evade and/or subvert this pathway (reviewed in references 9, 11, 14, 35, 37, 60, 61, and 77). Several members of the herpesvirus family, most notably herpes simplex virus type 1, inhibit autophagy within an infected cell and encode proteins that block and/or target intracellular signaling pathways that regulate autophagy (reviewed in references 60 and 61). However, some viruses not only evade autophagy but also appear to take advantage of the process; several RNA viruses induce autophagy and exploit the pathway during their replication (1, 12, 15, 31, 40, 43, 76, 93, 96). Viruses belonging to the Picornaviridae family and the Nidovirales order replicate their genomes on double-membraned vesicles that resemble autophagosomes; these vesicles are notably smaller in size than cellular autophagosomes and are decorated with proteins derived from the autophagic pathway (19, 21, 31, 37, 67, 68, 71, 92). Viral proteins encoded by poliovirus and equine arterivirus can trigger the formation of these autophagy-like vesicles (79, 80), and the expression of a single poliovirus protein, 2BC, is sufficient to induce lipidation of the host autophagy protein light chain 3 (LC3), encoded by the Atg8 gene (87). Taken together, these studies suggest that some viruses subvert the autophagy pathway to generate double-membraned vesicles that provide a surface for RNA replication (8, 37, 88). In addition, these vesicles may permit newly formed virions to escape from infected cells via a nonlytic route (36, 85).Although studies have demonstrated that the autophagic pathway may play an important role in virus infection in vitro, either to promote or to restrict viral replication, we are just beginning to appreciate and understand the function and effects of autophagy for virus infections in vivo. Autophagy acts in an antiviral fashion to limit tobacco mosaic virus replication and programmed cell death in plants (46), to prevent a pathogenic infection with vesicular stomatitis virus in flies (73), and to protect against fatal encephalitis in Sindbis virus- or herpes simplex virus type 1-infected mice (45, 59, 63). Nonetheless, to date there is a dearth of in vivo studies; animal models of virus infection are needed in order to better define the antiviral role of autophagy in vivo (41, 62). In addition, studies that address the role of viral subversion of autophagy in vivo are warranted. Does this process occur within infected animals, and is it required for viral replication in particular cell types or for viral pathogenesis? Recent studies have shown that autophagy not only promotes the replication of hepatitis B virus and enterovirus 71 in vitro but also may be induced by infection in vivo, potentially to benefit the virus rather than the host (28, 78).Type B coxsackieviruses (CVBs) are members of the Picornaviridae family and Enterovirus genus and, as such, are closely related to polioviruses. CVBs are important human pathogens that often induce severe acute and chronic diseases and cause morbidity and mortality (69, 91). CVBs are the most common cause of infectious myocarditis (38, 82) and frequently trigger pancreatitis and aseptic meningitis (7, 16, 29, 51). Tissue culture studies (93) have shown that CVB type 3 (CVB3) promotes LC3 conversion and autophagosome accumulation in virus-infected cells in vitro and that modulation of the autophagic pathway (using chemicals or small interfering RNA-mediated knockdown) to enhance or dampen autophagy results in an increase and a decrease, respectively, in viral protein expression and/or viral titers; however, the reported changes in viral titers were modest (2- to 4-fold). In the present study, we examine whether CVB3 activates the autophagic pathway in vivo, specifically in pancreatic acinar cells, which are a natural primary target for this virus. Using a mouse model of CVB3 infection, which faithfully recapitulates most aspects of CVB disease in humans, we demonstrate that this virus triggers LC3 conversion and also modulates other components of the autophagy machinery. In addition, using a recombinant CVB3 (rCVB3) that expresses Discosoma sp. red fluorescent protein (DsRed-CVB3), we identify virus-infected cells in situ and show that CVB3 infection increases autophagosome abundance in vivo. Lysosomal-associated membrane protein 1 (LAMP-1) immunostaining confirmed that amphisomes are generated in virus-infected cells but that autophagic flux was not substantially enhanced as the infection progressed; rather, there appears to be a substantial blockade in fusion with lysosomes. Finally, transmission electron microscopy (TEM) ultrastructural analysis of the infected pancreas confirmed that double-membraned autophagy-like vesicles as well as very large autophagic compartments (for which we have coined the term “megaphagosomes”) were generated in acinar cells following virus infection. Overall, these data provide compelling evidence that CVB3 induces autophagy in vivo and suggest that this picornavirus may subvert this process in a mammalian host.     

Rotavirus Infection Induces Transient Pancreatic Involution and Hyperglycemia in Weanling Mice

Rotavirus is a ubiquitous double-stranded RNA virus responsible for most cases of infantile gastroenteritis. It infects pancreatic islets in vitro and is implicated as a trigger of autoimmune destruction of islet beta cells leading to type 1 diabetes, but pancreatic pathology secondary to rotavirus infection in vivo has not been documented. To address this issue, we inoculated 3 week-old C57Bl/6 mice at weaning with rhesus rotavirus, which is closely related to human rotaviruses and known to infect mouse islets in vitro. Virus was quantified in tissues by culture-isolation and enzyme-linked immunosorbent assay. A requirement for viral double stranded RNA was investigated in toll-like receptor 3 (TLR3)-deficient mice. Cell proliferation and apoptosis, and insulin expression, were analyzed by immunohistochemistry. Following rotavirus inoculation by gavage, two phases of mild, transient hyperglycemia were observed beginning after 2 and 8 days. In the first phase, widespread apoptosis of pancreatic cells was associated with a decrease in pancreas mass and insulin production, without detectable virus in the pancreas. These effects were mimicked by injection of the double-stranded RNA mimic, polyinosinic-polycytidylic acid, and were TLR3-dependent. By the second phase, the pancreas had regenerated but islets were smaller than normal and viral antigen was then detected in the pancreas for several days. These findings directly demonstrate pathogenic effects of rotavirus infection on the pancreas in vivo, mediated initially by the interaction of rotavirus double-stranded RNA with TLR3.     

INF1 Elicitin Activates Jasmonic Acid- and Ethylene-mediated Signalling Pathways and Induces Resistance to Bacterial Wilt Disease in Tomato

Phytophthora infestans , the cause of potato and tomato late blight disease, produces INF1 elicitin, a 10 kDa extracellular protein. INF1 induces a hypersensitive response (HR) and systemic acquired resistance in species of the Nicotiana genus and a few other genera. We analysed the response of tomato to INF1 and INF1 S3 , which has a Cys to Ser substitution at position 3 of the processed protein and therefore lacks HR induction activity in tobacco. No HR cell death was induced in either INF1- or INF1 S3 -treated tomato leaves. The expression of salicylic acid (SA)-responsive PR-1a ( P6 ) and PR-2a genes was not induced by treatment with either INF1 or INF1 S3 . However, the expression of jasmonic acid (JA)-responsive PR-6 encoding proteinase inhibitor II, LeATL6 encoding ubiquitin ligase E3, and LOX-E encoding lipoxygenase, was up-regulated in tomato leaves treated with INF1 but not in those treated with INF1 S3 . Their induction was completely compromised in INF1-treated jai1-1 mutant tomato, in which the JA signalling pathway is impaired. The accumulation of ethylene (ET) and the expression of ET-responsive genes were also induced in tomato by INF1 but not INF1 S3 treatment. The activation of JA and ET-mediated signals but not the SA-mediated signalling in INF1-treated tomato was also demonstrated by global gene expression analysis. INF1-treated tomatoes, but not those treated with INF1 S3 , exhibited resistance to bacterial wilt disease caused by Ralstonia solanacearum . Thus, INF1 seems to induce resistance to bacterial wilt disease in tomato and activate JA- and ET-mediated signalling pathways without development of HR cell death.     

Mast Cell Chymase Degrades the Alarmins Heat Shock Protein 70, Biglycan,HMGB1, and Interleukin-33 (IL-33) and Limits Danger-induced Inflammation

During infection and tissue damage, virulence factors and alarmins are pro-inflammatory and induce activation of various immune cells including macrophages and mast cells (MCs). Activated MCs instantly release preformed inflammatory mediators, including several proteases. The chymase mouse mast cell protease (MCPT)-4 is thought to be pro-inflammatory, whereas human chymase also degrades pro-inflammatory cytokines, suggesting that chymase instead limits inflammation. Here we explored the contribution of MCPT4 and human chymase to the control of danger-induced inflammation. We found that protein extracts from wild type (WT), carboxypeptidase A3-, and MCPT6-deficient mice and MCs and recombinant human chymase efficiently degrade the Trichinella spiralis virulence factor heat shock protein 70 (Hsp70) as well as endogenous Hsp70. MC-(W^sash)-, serglycin-, NDST2-, and MCPT4-deficient extracts lacked this capacity, indicating that chymase is responsible for the degradation. Chymase, but not MC tryptase, also degraded other alarmins, i.e. biglycan, HMGB1, and IL-33, a degradation that was efficiently blocked by the chymase inhibitor chymostatin. IL-7, IL-22, GM-CSF, and CCL2 were resistant to chymase degradation. MCPT4-deficient conditions ex vivo and in vivo showed no reduction in added Hsp70 and only minor reduction of IL-33. Peritoneal challenge with Hsp70 resulted in increased neutrophil recruitment and TNF-α levels in the MCPT4-deficient mice, whereas IL-6 and CCL2 levels were similar to the levels found in WT mice. The rapid and MC chymase-specific degradation of virulence factors and alarmins may depend on the presence of accessible extended recognition cleavage sites in target substrates and suggests a protective and regulatory role of MC chymase during danger-induced inflammation.     

Dicer Is Required for Maintenance of Adult Pancreatic Acinar Cell Identity and Plays a Role in Kras-Driven Pancreatic Neoplasia

The role of miRNA processing in the maintenance of adult pancreatic acinar cell identity and during the initiation and progression of pancreatic neoplasia has not been studied in detail. In this work, we deleted Dicer specifically in adult pancreatic acinar cells, with or without simultaneous activation of oncogenic Kras. We found that Dicer is essential for the maintenance of acinar cell identity. Acinar cells lacking Dicer showed increased plasticity, as evidenced by loss of polarity, initiation of epithelial-to-mesenchymal transition (EMT) and acinar-to-ductal metaplasia (ADM). In the context of oncogenic Kras activation, the initiation of ADM and pancreatic intraepithelial neoplasia (PanIN) were both highly sensitive to Dicer gene dosage. Homozygous Dicer deletion accelerated the formation of ADM but not PanIN. In contrast, heterozygous Dicer deletion accelerated PanIN initiation, revealing complex roles for Dicer in the regulation of both normal and neoplastic pancreatic epithelial identity.     

Effects of Dominant/Subordinate Social Status on Formalin-Induced Pain and Changes in Serum Proinflammatory Cytokine Concentrations in Mice

Current investigations regarding social stress primarily focus on the health consequences of being in stressful social hierarchies. The repetitive nature of social conflicts seems to favor an induction of hyperalgesia or hypoalgesia, both in rodents and humans. Additionally, social conflicts may affect the immune system. In order to better establish the pain and immune responses to stress, the present study implemented a sensory contact model on 32 male BALB/c mice. Subsequent to establishing a dominance/submissive social relationship, each mouse was injected with formalin (20 μl, 2%) and their pain behavior was scored and serum concentrations of proinflammatory cytokines IL-1 and IL-6, and corticosterone were also measured. Test results revealed that subordinate mice were hypoalgesic during chronic phase of formalin test compared to control and dominant mice (P<0.05). On the other hand, subordinate mice were hyperalgesic compared to dominant mice during the whole acute phase of formalin test (P<0.05). Corticosterone, IL-1 and IL-6 concentrations were much higher in serum of dominant and subordinate mice than in the control group (p<0.05). The results indicated that, although both dominant and subordinate animals displayed an increase in serum corticosterone and proinflammatory cytokines during social interactions, their response to pain perception differently was affected with the social status.     

Subversion of Autophagy in Adherent Invasive Escherichia coli-Infected Neutrophils Induces Inflammation and Cell Death

Invading bacteria are recognized, captured and killed by a specialized form of autophagy, called xenophagy. Recently, defects in xenophagy in Crohn’s disease (CD) have been implicated in the pathogenesis of human chronic inflammatory diseases of uncertain etiology of the gastrointestinal tract. We show here that pathogenic adherent-invasive Escherichia coli (AIEC) isolated from CD patients are able to adhere and invade neutrophils, which represent the first line of defense against bacteria. Of particular interest, AIEC infection of neutrophil-like PLB-985 cells blocked autophagy at the autolysosomal step, which allowed intracellular survival of bacteria and exacerbated interleukin-8 (IL-8) production. Interestingly, this block in autophagy correlated with the induction of autophagic cell death. Likewise, stimulation of autophagy by nutrient starvation or rapamycin treatment reduced intracellular AIEC survival and IL-8 production. Finally, treatment with an inhibitor of autophagy decreased cell death of AIEC-infected neutrophil-like PLB-985 cells. In conclusion, excessive autophagy in AIEC infection triggered cell death of neutrophils.     

Liraglutide Improves Pancreatic Beta Cell Mass and Function in Alloxan-Induced Diabetic Mice

Glucagon-like peptide-1 (GLP-1) receptor agonists potentiate glucose-induced insulin secretion. In addition, they have been reported to increase pancreatic beta cell mass in diabetic rodents. However, the precise mode of action of GLP-1 receptor agonists still needs to be elucidated. Here we clarify the effects of the human GLP-1 analog liraglutide on beta cell fate and function by using an inducible Cre/loxP-based pancreatic beta cell tracing system and alloxan-induced diabetic mice. Liraglutide was subcutaneously administered once daily for 30 days. The changes in beta cell mass were examined as well as glucose tolerance and insulin secretion. We found that chronic liraglutide treatment improved glucose tolerance and insulin response to oral glucose load. Thirty-day treatment with liraglutide resulted in a 2-fold higher mass of pancreatic beta cells than that in vehicle group. Liraglutide increased proliferation rate of pancreatic beta cells and prevented beta cells from apoptotic cells death. However, the relative abundance of YFP-labeled beta cells to total beta cells was no different before and after liraglutide treatment, suggesting no or little contribution of neogenesis to the increase in beta cell mass. Liraglutide reduced oxidative stress in pancreatic islet cells of alloxan-induced diabetic mice. Furthermore, the beneficial effects of liraglutide in these mice were maintained two weeks after drug withdrawal. In conclusion, chronic liraglutide treatment improves hyperglycemia by ameliorating beta cell mass and function in alloxan-induced diabetic mice.     

Sulforaphane Induces Cell Cycle Arrest and Apoptosis in Acute Lymphoblastic Leukemia Cells

Acute lymphoblastic leukemia (ALL) is the most common hematological cancer in children. Although risk-adaptive therapy, CNS-directed chemotherapy, and supportive care have improved the survival of ALL patients, disease relapse is still the leading cause of cancer-related death in children. Therefore, new drugs are needed as frontline treatments in high-risk disease and as salvage agents in relapsed ALL. In this study, we report that purified sulforaphane, a natural isothiocyanate found in cruciferous vegetables, has anti-leukemic properties in a broad range of ALL cell lines and primary lymphoblasts from pediatric T-ALL and pre-B ALL patients. The treatment of ALL leukemic cells with sulforaphane resulted in dose-dependent apoptosis and G2/M cell cycle arrest, which was associated with the activation of caspases (3, 8, and 9), inactivation of PARP, p53-independent upregulation of p21^CIP1/WAF1, and inhibition of the Cdc2/Cyclin B1 complex. Interestingly, sulforaphane also inhibited the AKT and mTOR survival pathways in most of the tested cell lines by lowering the levels of both total and phosphorylated proteins. Finally, the administration of sulforaphane to the ALL xenograft models resulted in a reduction of tumor burden, particularly following oral administration, suggesting a potential role as an adjunctive agent to improve the therapeutic response in high-risk ALL patients with activated AKT signaling.     

A Novel Burdock Fructooligosaccharide Induces Changes in the Production of Salicylates, Activates Defence Enzymes and Induces Systemic Acquired Resistance to Colletotrichum orbiculare in Cucumber Seedlings

The ability of burdock fructooligosaccharide (BFO), a type of linear fructooligosaccharide extracted and isolated from the roots of Arctium lappa , to induce systemic acquired resistance (SAR) was studied in cucumber seedlings. BFO strongly induced changes in salicylic acid (SA) and SA-glucoside (SAG) in BFO-treated leaves, and similar changes of SA and SAG were also found in untreated leaves of the same seedling. The level of SA in the first leaves sprayed with BFO (5.0 g/l) increased by 3.6 times after 24 h and then gradually declined from 48 to 96 h and finally decreased to a nadir at 120 h. The SAG level increased by 2.1 times at 24 h and then continued to increase to about 10.0 times as much as that in control from 96 to 120 h. The levels of SA in the untreated leaves of the same seedling only increased by 1.6–1.9 times during the period of 24–72 h followed by a decrease at 120 h, while SAG increased by 1.1 times at 24 h but steadily continued to increase to its maximum from 24 to 120 h. In summary, the patterns of expression of SA and SAG in the untreated leaf were similar to that of the treated leaf of the same seedling, while the pattern of expression of SAG was quite different from that of SA both in the treated and untreated leaves. Pretreatment with BFO reduced the lesions caused by Colletotrichum orbiculare by 56.8%. Additionally, the amount of lignin and the activities of some defensive enzymes including peroxidase, superoxide dismutase, polyphenoloxidase and β-1,3-glucanase significantly increased in the first leaves pretreated with BFO and followed with C. orbiculare inoculation. These results demonstrate that BFO can enhance the contents of endogenous SA, the resistance against C. orbiculare , and the activities of defensive enzymes of cucumber seedlings.     

A Novel Mucosal Vaccine Against Foot-and-Mouth Disease Virus Induces Protection in Mice and Swine

Epitopes of a foot-and-mouth disease virus (FMDV) capsid protein VP1 complex and a chimera of 6×His-tagged cholera toxin B subunit (hCTB) were expressed in Hansenula polymorpha and used together as a mucosal vaccine. Antibody and cytokine responses to VP1–hCTB vaccine and protection against FMDV were evaluated by ELISA and a virus challenge test in mice, respectively. VP1–hCTB directly enhanced the expression of interleukin-5 (IL-5) both in serum and supernatants of cultured spleen cells. After challenging suckling mice with 10⁵ FMDV (=50% lethal dosage per mouse) a greater protection was seen after intraperitoneal and intranasal vaccinations than after oral vaccination. In swine immunized with VP1–hCTB, immune responses were achieved after three administrations, and the vaccine protected swine (80%) when challenged with 10^6.5 FMDV (=50% infectious dosage per swine). These results demonstrated the possibility of using CTB as a mucosal adjuvant to elicit protective immune responses against FMDV. Houhui Song, Zhiliang Wang and Dongxia Zheng contributed equally to this work.     

T Cell Activation and Proinflammatory Cytokine Production in Clinically Cured Tuberculosis Are Time-Dependent and Accompanied by Upregulation of IL-10

Background

Th1 cytokines are essential for the control of M. tuberculosis infection. The role of IL-10 in tuberculosis is controversial and there is an increasing body of evidence suggesting that the relationship between Th1 cytokines and IL-10 is not as antagonistic as it was first believed, and that these cytokines may complement each other in infectious diseases.

Methods

The present study evaluated the activating capacity of CD4+ and CD8+ T cell repertoire in response to antigen stimulation through the expression of CD69 using Flow Cytometry, as well as the functionality of PBMCs by determining the cytokine profile in patients with active tuberculosis and in clinically cured patients after in vitro stimulation using ELISA. Treated patients were subdivided according to time after clinical cure (<12 months or >12 months post-treatment).

Results

We observed that T cell activation was higher in TB-treated patients, especially CD8+ T cell activation in TB-Treated >1 year. Th1 cytokines were significantly higher in TB-Treated, and the levels of IFN-γ and TNF-α increased continuously after clinical cure. Moreover, IL-10 production was significantly higher in cured patients and it was also enhanced in cured patients over time after treatment. Th17, Th2 and Th22 cytokines showed no statistically significant differences between Healthy Donors, Active-TB and TB-Treated.

Conclusions

This study describes a scenario in which potentiation of CD4+ and CD8+ T cell activation and increased Th1 cytokine production are associated with the clinical cure of tuberculosis in the absence of significant changes in Th2 cytokine production and is accompanied by increased production of IL-10. In contrast to other infections with intracellular microorganisms, this response occurs later after the end of treatment.     

Chlamydia pneumoniae Infection in Mice Induces Chronic Lung Inflammation,iBALT Formation,and Fibrosis

Chlamydia pneumoniae (CP) lung infection can induce chronic lung inflammation and is associated with not only acute asthma but also COPD exacerbations. However, in mouse models of CP infection, most studies have investigated specifically the acute phase of the infection and not the longer-term chronic changes in the lungs. We infected C57BL/6 mice with 5×10⁵ CP intratracheally and monitored inflammation, cellular infiltrates and cytokine levels over time to investigate the chronic inflammatory lung changes. While bacteria numbers declined by day 28, macrophage numbers remained high through day 35. Immune cell clusters were detected as early as day 14 and persisted through day 35, and stained positive for B, T, and follicular dendritic cells, indicating these clusters were inducible bronchus associated lymphoid tissues (iBALTs). Classically activated inflammatory M1 macrophages were the predominant subtype early on while alternatively activated M2 macrophages increased later during infection. Adoptive transfer of M1 but not M2 macrophages intratracheally 1 week after infection resulted in greater lung inflammation, severe fibrosis, and increased numbers of iBALTS 35 days after infection. In summary, we show that CP lung infection in mice induces chronic inflammatory changes including iBALT formations as well as fibrosis. These observations suggest that the M1 macrophages, which are part of the normal response to clear acute C. pneumoniae lung infection, result in an enhanced acute response when present in excess numbers, with greater inflammation, tissue injury, and severe fibrosis.