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B lymphocyte-mediated humoral immune response is essential for protection against infectious diseases. Deeper research in B cell biology, particularly metabolism is required for the better understanding of its properties in homeostasis and in diseases. Emerging immunometabolism, including anabolism and catabolism, has tremendously impacts on immune cells from development to function and markedly advances our view on immunoregulation. Growing evidence suggests that the ultimate effect of intracellular metabolism on immune cell functions is not only influenced by the external stimuli but also by the balance of the different metabolic pathways. However, B cell immunometabolism is not deeply investigated like T cells. The complex development and differentiation processes of B cell subsets have left many untouched, but fundamental aspects in B cell metabolism. Available evidence demonstrated that the intracellular metabolism has the ubiquitous impact on B cell fate and function decisions at the transcriptional regulation and signal transduction processes. In this review, we update the recent development in the immunometabolism of B cells with the latest findings including the immune-metabolic steering on B cell development, differentiation, and function skewing, and emphasis on how immunometabolism landscape may shape B cell functions in metabolic, autoimmune, and inflammatory disorders. The metabolic interaction of B cells with other immune cells in disease context will also be discussed.

Adherent-invasiveEscherichia coli(AIEC) have been implicated in the aetiology of Crohn’s Disease (CD). They are characterized by an ability to adhere to and invade intestinal epithelial cells, and to replicate intracellularly in macrophages resulting in inflammation. Proline-rich tyrosine kinase 2 (PYK2) has previously been identified as a risk locus for inflammatory bowel disease through genome wide association studies. It is overexpressed in patients with colorectal cancer, a major long-term complication of CD. Here we show that Pyk2 levels are significantly increased during AIEC infection of murine macrophages while an inhibitor of Pyk2, PF-431396 hydrate, significantly decreased intramacrophage AIEC numbers. Imaging flow cytometry indicated that Pyk2 inhibition blocked intramacrophage replication of AIEC with no change in the overall number of infected cells, but a significant reduction in bacterial burden per cell. This reduction in intracellular bacteria resulted in a 20-fold decrease in tumour necrosis factor α secretion by cells post-AIEC infection. These data demonstrate a key role for Pyk2 in modulating AIEC intracellular replication and associated inflammation and may provide a new avenue for future therapeutic intervention in CD.

Adult fibrosarcoma is an aggressive subtype of soft tissue sarcoma (STS), in which high expression of KIF20A indicates a poor prognosis. However, the precise role of KIF20A in fibrosarcoma progression remains unknown. In this study, we initially examined KIF20A expression and function in the human fibrosarcoma cell line HT-1080. The results showed that KIF20A was highly expressed in HT-1080, knockdown of KIF20A impaired cell proliferation, migration, invasion and induced G2/M arrest and cell apoptosis. Transcriptome study suggested that PI3K-Akt signal pathway was involved in these biological changes. We confirmed that PI3K-Akt and NF-κB signaling pathways were impaired after the down-regulation of KIF20A, which can be reversed by the Akt activator SC79 in HT-1080 in vitro. In a xenograft mouse model, knockdown of KIF20A inhibited tumor growth, Ki67 expression and liver metastasis. Taken together, our results suggested that KIF20A promoted fibrosarcoma progression via PI3K-Akt signaling pathway and might be a potential therapeutic target for fibrosarcoma.

Increasing evidence suggests that IL-33 plays an important role in regulating tumor development. However, conflicting results, obtained from numerous studies, have highlighted the divergent functions of IL-33. The detailed mechanisms by which IL-33 modulates tumor development merit further investigation. Here, we report that IL-33 administration can effectively inhibit the development of pulmonary metastasis of breast cancer in a mouse. In our model, IL-33 promotes the production of TNF-α by macrophages, which increases IL-33 specific receptor (ST2) expression on natural killer (NK) cells and is pivotal in IL-33-induced NK cell activation. IL-33 treatment also facilitates the production of CCL5 in the lung by eosinophils and CD8+ T cells, which mediates the recruitment of NK cells to the tumor microenvironment. The systemic activation and local recruitment of NK cells result in potent tumor rejection in the lung. Our study reports a novel mechanism for the IL-33-meditated suppression of metastatic cancer and provides potential therapeutic strategies for targeting metastatic tumor.

The emergence of the novel coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to millions of infections and is exerting an unprecedented impact on society and economies worldwide. The evidence showed that heart failure (HF) is a clinical syndrome that could be encountered at different stages during the progression of COVID-19. Shenfu injection (SFI), a traditional Chinese medicine (TCM) formula has been widely used for heart failure therapy in China and was suggested to treat critical COVID-19 cases based on the guideline for diagnosis and treatment of COVID-19 (the 7th version) issued by National Health Commission of the People's Republic of China. However, the active components, potential targets, related pathways, and underlying pharmacology mechanism of SFI against COVID-19 combined with HF remain vague.To investigate the effectiveness and possible pharmacological mechanism of SFI for the prevention and treatment of COVID-19 combined with HF.In the current study, a network analysis approach integrating active compound screening (drug-likeness, lipophilicity, and aqueous solubility models), target fishing (Traditional Chinese Medicine Systems Pharmacology, fingerprint-based Similarity Ensemble Approach, and PharmMapper databases), compound-target-disease network construction (Cytoscape software), protein-protein interaction network construction (STRING and Cytoscape software), biological process analysis (STRING and Cytoscape plug-in Clue GO) and pathway analysis (Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis) was developed to decipher the active ingredients, potential targets, relevant pathways, and the therapeutic mechanisms of SFI for preventing and treating COVID-19 combined with HF.Finally, 20 active compounds (DL ≥ 0.18, 1≤Alog P ≤ 5, and -5≤LogS ≤ -1) and 164 relevant targets of SFI were identified related to the development of COVID-19 combined with HF, which were mainly involved in three biological processes including metabolic, hemostasis, and cytokine signaling in immune system. The C-T-D network and reactome pathway analysis indicated that SFI probably regulated the pathological processes of heart failure, respiratory failure, lung injury, and inflammatory response in patients with COVID-19 combined with HF through acting on several targets and pathways. Moreover, the venn diagram was used to identify 54 overlapped targets of SFI, COVID-19, and HF. KEGG pathway enrichment analysis showed that 54 overlapped targets were highly enriched to several COVID-19 and HF related pathways, such as IL-17 signaling pathway, Th17 cell differentiation, and NF-kappa B signaling pathway.A comprehensive network analysis approach framework was developed to systematically elucidate the potential pharmacological mechanism of SFI for the prevention and treatment of SFI against COVID-19 combined with HF. The current study may not only provide in-depth understanding of the pharmacological mechanisms of SFI, but also a scientific basis for the application of SFI against COVID-19 combined with HF.

Background Environmental pollutants, which coexist with allergens, have been associated with the exacerbation of asthma. However, the underlying molecular mechanisms remain elusive. We sought to determine whether benzo(a)pyrene (BaP) co‐exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1‐induced asthma and its underlying mechanisms. Methods The effect of BaP was investigated in Der f 1‐induced mouse model of asthma, including airway hyper‐responsiveness, allergic inflammation, and epithelial‐derived cytokines. The impact of BaP on Der f 1‐induced airway epithelial cell oxidative stress (ROS) and cytokine release was further analyzed. The role of aryl hydrocarbon receptor (AhR) signaling in BaP‐promoted Der f 1‐induced ROS, cytokine production, and allergic inflammation was also investigated. Results Compared with Der f 1, BaP co‐exposure with Der f 1 led to airway hyper‐responsiveness and increased lung inflammation in mouse model of asthma. Increased expression of TSLP, IL‐33, and IL‐25 was also found in the airways of these mice. Moreover, BaP co‐exposure with Der f 1 activated AhR signaling with increased expression of AhR and CYP1A1 and promoted airway epithelial ROS generation and TSLP and IL‐33, but not IL‐25, expression. Interestingly, AhR antagonist CH223191 or cells with AhR knockdown abrogated the increased expression of ROS, TSLP, and IL‐33. Furthermore, ROS inhibitor N‐acetyl‐L‐cysteine (NAC) also suppressed BaP co‐exposure‐induced expression of epithelial TSLP, IL‐33, and IL‐25. Finally, AhR antagonist CH223191 and NAC inhibited BaP co‐exposure with Der f 1‐induced lung inflammation. Conclusions Our findings suggest that BaP facilitates Der f 1‐induced epithelial cytokine release through the AhR‐ROS axis., Compared with Der f 1 alone, BaP and Der f 1 co‐exposure leads to airway hyperresponsiveness and increases Th2‐associated lung inflammation in mouse model of asthma. AhR signaling plays a critical role in BaP and Der f 1 co‐exposure‐induced oxidative stress and cytokine expression in airway epithelial cells. The AhR‐ROS axis regulates BaP and Der f 1 co‐exposure‐induced epithelial cytokine expression and allergic inflammation. AhR: Aryl hydrocarbon receptor; BaP: benzo(a)pyrene; CLRs: C‐type lectin receptors; HDM: house dust mite; PAR2: protease‐activated receptor 2; ROS: reactive oxygen species.

Background: We have previously demonstrated that benzo(a)pyrene (BaP) co-exposure with dermatophagoides group 1 allergen (Der f 1) can potentiate Der f 1-induced airway inflammation. We sought to investigate the molecular mechanisms underlying the potentiation of BaP exposure on Der f 1-induced airway inflammation. Methods: BaP co-exposure with Der f 1-induced activation of TGFβ1 signaling was analyzed in airway epithelial cells (HBECs) and in asthma mouse model. The role of aryl hydrocarbon receptor (AhR) and RhoA in BaP co-exposure-induced TGFβ1 signaling was investigated. AhR binding sites in RhoA were predicted and experimentally confirmed by luciferase reporter assays. The role of RhoA in BaP co-exposure-induced airway hyper-responsiveness (AHR) and allergic inflammation was examined. Results: BaP co-exposure potentiates Der f 1-induced TGFβ1 signaling activation in HBECs and in the airways of asthma mouse model. The BaP co-exposure-induced the activation of TGFβ1 signaling was attenuated by either AhR antagonist CH223191 or AhR knockdown in HBECs. Furthermore, AhR knockdown led to the reduction of BaP co-exposure-induced active RhoA. Inhibition of RhoA signaling with fasudil, a RhoA/ROCK inhibitor, suppressed BaP co-exposure-induced TGFβ1 signaling activation. This was further confirmed in HBECs expressing constitutively active RhoA (RhoA-L63) or dominant negative RhoA (RhoA-N19). Luciferase reporter assays showed prominently increased promoter activities for the AhR binding sites in the promoter region of RhoA. Inhibition of RhoA suppressed co-exposure-induced AHR, Th2-associated airway inflammation and TGFβ1 signaling activation in asthma. Conclusions: Our studies identified a functional axis of AhR-RhoA that regulates TGFβ1 signaling activation, leading to allergic airway inflammation and asthma.

Interleukin-33 has been demonstrated to be associated with liver damage. However, its potential value in hepatitis B virus (HBV) infection remains unknown. This study was designed to investigate the role of IL-33 in hydrodynamic HBV mouse model. Different doses of IL-33 were used to treat HBV wild-type, ST2 knockout, CD8+ T depletion, NK depletion C57BL/6 mice and C.B-17 SCID and nod SCID mouse, respectively. The concentrations of HBV DNA, HBsAg, HBeAg, and molecules related to liver function were detected in the collected serum at different time points from model mice. Intrahepatic HBcAg was visualized by immunohistochemical staining of liver tissues. In vitro, hepG2 cells were transfected with pAAV-HBV 1.2, then treated with IL-33. The results showed that IL-33 significantly reduced HBV DNA and HBsAg in a dose-dependent manner in HBV wild-type mice. However, in the IL-33 specific receptor ST2 knockout mice, their antiviral effects could not be exerted. Through immunodeficient animal models and in vivo immune cell depletion mouse model, we found that IL-33 could not play antiviral effects without NK cells. Moreover, IL-33 could reduce the levels of HBsAg and HBeAg in the supernatant of HBV-transfected hepG2 cells in vitro. Our study revealed that IL-33 could inhibit HBV through ST2 receptor in the HBV mouse model, and this effect can be impaired without NK cell. Additionally, IL-33 had the direct anti-HBV effect in vitro, indicating that IL-33 could be a potent inducer of HBV clearance and a promising drug candidate.

The monophosphoryl lipid A (MPLA) is a detoxified LPS derivative and an emerging safe immune adjuvant in human vaccine development. The adjuvant MPLA promotes antigen-presenting cell (APC) function and preferentially induces a Th1 response following vaccination. However, the mechanism by which the MPLA detoxicates and exerts its adjuvants effect on T-cell, particualrly the Th1 response is unknown.We assessed the direct effects of MPLA on murine and human CD4We report that CD4These data provide evidence, at least in part, for the safe induction of an appropriate level of Th1 response by adjuvant MPLA in human vaccine development.

Background The monophosphoryl lipid A (MPLA) is a detoxified LPS derivative and an emerging safe immune adjuvant in human vaccine development. The adjuvant MPLA promotes antigen-presenting cell (APC) function and preferentially induces a Th1 response following vaccination. However, the mechanism by which the MPLA detoxicates and exerts its adjuvants effect on T-cell, particualrly the Th1 response is unknown. Aims We assessed the direct effects of MPLA on murine and human CD4+ T-cell proliferation and the profile of cytokine production ex vivo. Results We report that CD4+ T-cells only express functional TLR2 and TLR4 when activated by TCR stimulation, in particularly in the presence of IFNα. The activated T cells thereafter can respond directly to MPLA. MPLA does not affect T-cell proliferation in human T cells, but can induce a balanced Th1 cytokine profile in CD4+ T-cells by reducing the production of Th1 cytokines and enhancing the production of the regulatory cytokine IL-10. The MPLA-mediated regulatory effect on activated CD4+ T-cells is TLR2 and TLR4 dependent and can be abolished by the lipid A blocker polymyxin B. Conclusion These data provide evidence, at least in part, for the safe induction of an appropriate level of Th1 response by adjuvant MPLA in human vaccine development.

Toll-like receptors (TLRs) 2 and 4 recognize different endogenous and exogenous agonists and play a distinct role in infection and inflammation. However, their ultimate effect in a given infectious and inflammatory disease is less understood. We produced polyclonal anti-murine TLR2 and TLR4 antibodies and investigated their effect on cutaneous leishmaniasis and inflammatory arthritis. Administration of these antibodies to susceptible BALB/c mice, infected in the footpad with Leishmania major, reduced footpad swelling but not the parasite load compared with mice treated with control IgG. The antibodies synergistically reduced leishmanial-specific T-cell proliferation, T helper type 1 and type 2 cytokine production, specific IgG1 and IgG2a antibody synthesis, and T-cell receptor and co-stimulatory molecule expression on dendritic cells in infected mice. We then tested the effect of these antibodies on collagen-induced arthritis (CIA) in DBA/1 mice, a classic model of chronic inflammation. Both antibodies markedly suppressed the development of clinical parameters with concomitant reduction of pro-inflammatory cytokine production. These data therefore suggest that anti-TLR2 and 4 antibodies may have a synergistic therapeutic effect on inflammatory disease in vivo.

Regulatory B cells (Breg) have attracted increasing attention for their roles in maintaining peripheral tolerance. Interleukin 33 (IL-33) is a recently identified IL-1 family member, which leads a double-life with both pro- and anti-inflammatory properties. We report here that peritoneal injection of IL-33 exacerbated inflammatory bowel disease in IL-10-deficient (IL-10−/−) mice, whereas IL-33-treated IL-10-sufficient (wild type) mice were protected from the disease induction. A phenotypically unconventional subset(s) (CD19+CD25+CD1dhiIgMhiCD5-CD23-Tim-1-) of IL-10 producing Breg-like cells (BregIL-33) was identified responsible for the protection. We demonstrated further that BregIL-33 isolated from these mice could suppress immune effector cell expansion and functions and, upon adoptive transfer, effectively blocked the development of spontaneous colitis in IL-10−/− mice. Our findings indicate an essential protective role, hence therapeutic potential, of BregIL-33 against mucosal inflammatory disorders in the gut., Highlights • IL-33, a branded ‘Th2’ cytokine, could accelerate the Th1-mediated colitis under an IL-10 deficient condition. • IL-10 sufficient mice were protected from the IL-33-mediated mucosal inflammation. • IL-33 induced a phenotypically unique subset of IL-10-producing regulatory B cells (BregIL-33). • Adoptive transfer of BregIL-33 blocked the spontaneous colitis in IL-10-deficient mice (therapeutic potential). • Our findings offer new insights into the immunological mechanisms underlying mucosal inflammation, and its regulation.

Cigarette smoke (CS) is the principal cause of pulmonary inflammatory response. IL-28 (IFN-λ) is a novel group of class II cytokines targeting the epithelial cells and IL-28 responses prominent in lungs can exert important immunomodulatory effects. We tested the hypothesis that IL-28B may modulate the lung inflammation induced by CS. Groups of mice were exposed to CS two times per day for 11 consecutive days. CS exposure induced lymphocyte, neutrophil and macrophage infiltration and inflammatory cytokine (IL-1β, tumor necrosis factor-α (TNF)-α, IL-17, and IL-4) in the airways. More importantly, all these CS-induced pathogenic changes were significantly inhibited by hydrodynamic delivery of plasmid DNA encoding mouse IL-28B. Thus, our results suggest that IL-28 cytokines are beneficial for the suppression of CS-mediated airway inflammation and may be a therapeutic target in CS-related diseases.

Background and Purpose IL-33 signals through ST2 receptors and induces adaptive and innate inflammation. IL-33/ST2 is involved in adaptive inflammation-induced pain. Here, we have investigated the contribution of IL-33/ST2-triggered mechanisms to carrageenin-induced innate inflammation. Experimental Approach Carrageenin- and IL–33-induced inflammatory responses were assessed in BALB/c- (WT) and ST2-deficient (−/−) mice as follows: oedema (plethysmometer), myeloperoxidase activity (colorimetric assay), mechanical hyperalgesia (electronic version of von Frey filaments), cytokine levels (ELISA), PGE2 (RIA), mRNA expression (quantitative PCR), drug treatments targeting leukocyte recruitment (fucoidin), TNF-α (infliximab), CXCL1 (antibody to CXCL1), IL-1 (IL-1ra), endothelin ETA (clazosentan) and ETB (BQ788) receptors and COX (indomethacin). Key Results Carrageenin injection increased ST2 and IL-33 mRNA expression and IL-33 production in paw skin samples. Carrageenin-induced paw oedema, hyperalgesia and myeloperoxidase activity were reduced in ST2−/− compared with WT mice, effects mimicked by IL-33 injection in the paw. Furthermore, IL–33-induced hyperalgesia was reduced by fucoidin suggesting a role for recruited leukocytes in its hyperalgesic effect. IL–33-induced hyperalgesia in naive mice was reduced by treatments targeting TNF, CXCL1, IL-1, endothelin receptors and COX while carrageenin-induced ST2-dependent TNF-α, CXCL1, IL-1β, IL-10 and PGE2 production and preproET-1 mRNA expression. Combining IL-33 and carrageenin at doses that were ineffective as single treatment induced significant hyperalgesia, oedema, myeloperoxidase activity and cytokine production in a ST2-dependent manner. Conclusions and Implications IL-33/ST2 signalling triggers the production of inflammatory mediators contributing to carrageenin-induced inflammation. These data reinforces the importance of IL-33/ST2 signalling as a target in innate inflammation and inflammatory pain.

The mechanism by which cigarette smoke (CS) causes chronic obstructive pulmonary disease (COPD) is poorly understood. Interleukin-33 (IL-33) is a pleiotropic cytokine predominantly expressed in lung tissue and can elicit airway inflammation in naive mice. We tested the hypothesis that IL-33 is induced by CS and contributes to CS-mediated airway inflammation in a mouse model of CS-induced COPD. Groups of mice were exposed to CS three times per day for 4 consecutive days. The expression levels of IL-33 and ST2 were markedly enhanced in the lung tissue of mice inhaling CS. Exposure to CS also induced neutrophil and macrophage infiltration and expression of inflammatory cytokines (IL-1β, tumour necrosis factor-α, IL-17), chemokines (monocyte chemoattractant protein-1) and mucin 5, subtypes A and C in the airways. More importantly, all of these CS-induced pathogenic changes were significantly inhibited by treatment with neutralizing anti-IL-33 antibody delivered intranasally. Hence, our results suggest that IL-33 plays a critical role in CS-mediated airway inflammation and may be a therapeutic target in CS-related diseases, including COPD.

Neuropathic pain from injury to the peripheral and CNS represents a major health care issue. We have investigated the role of IL-33/IL-33 receptor (ST2) signaling in experimental models of neuropathic pain in mice. Chronic constriction injury (CCI) of the sciatic nerve induced IL-33 production in the spinal cord. IL-33/citrine reporter mice revealed that oligodendrocytes are the main cells expressing IL-33 within the spinal cord together with a minor expression by neurons, microglia. and astrocytes. CCI-induced mechanical hyperalgesia was reduced in IL-33R (ST2)(-/ -) mice compared with wild-type (WT) mice. Intrathecal treatment of WT mice with soluble IL-33 receptor (IL-33 decoy receptor) markedly reduced CCI-induced hyperalgesia. Consistent with these observations, intrathecal injection of IL-33 enhanced CCI hyperalgesia and induced hyperalgesia in naive mice. IL-33-mediated hyperalgesia during CCI was dependent on a reciprocal relationship with TNF-α and IL-1β. IL-33-induced hyperalgesia was markedly attenuated by inhibitors of PI3K, mammalian target of rapamycin, MAPKs (p38, ERK, and JNK), NF-κB, and also by the inhibitors of glial cells (microglia and astrocytes). Furthermore, targeting these signaling pathways and cells inhibited IL-33-induced TNF-α and IL-1β production in the spinal cord. Our study, therefore, reveals an important role of oligodendrocyte-derived IL-33 in neuropathic pain.

Background: The regulation and function of IgE in healthy individuals and in antigen-naive animals is not well understood. IL-33 administration increases serum IgE in mice with unknown mechanism. We tested the hypothesis that IL-33 provides an antigen-independent stimulus for IgE production and mast cell degranulation. Methods: IL-33 was administered to naive wild-type (WT), nude and ST2 / , IL-4 / , IL4Ra / and T-or B-cell-specific IL-4Ra / mice. IgEand cytokines were quantified by ELISA. T- and B-lymphocyte numbers and CD40L expression were determined by flow cytometry. Anaphylaxis was measured by temperature, mast cell degranulation and histamine release. Results: IL-33 enhanced IgE production in naive WT, T-IL-4Ra / but not in ST2 / , IL-4 / , IL-4Ra / or B-cell-specific IL-4Ra / mice, demonstrating IL-33 specificity and IL-4 dependency. Moreover, IL-4 was required for IL-33- induced B-cell proliferation and T-cell CD40L expression, which promotes IgE pro- duction. IL-33-induced IL-4 production was mainly from innate cells including mast cells and eosinophils. IL-33 increased mast cell surface IgE and triggered degranula- tion and systemic anaphylaxis in allergen-naive WT but not in IL-4Ra / mice. Conclusion: IL-33 amplifies IgE synthesis and triggers anaphylaxis in naive mice via IL-4, independent of allergen. IL-33 may play an important role in nonatopic allergy and idiopathic anaphylaxis.

Interleukin (IL)-33, a member of the IL-1 cytokine family, is an important modulator of the immune system associated with several immune-mediated disorders. High levels of IL-33 are expressed by the central nervous system (CNS) suggesting a potential role of IL-33 in autoimmune CNS diseases. We have investigated the expression and function of IL-33 in the development of experimental autoimmune encephalomyelitis (EAE) in mice. We report here that IL-33 and its receptor ST2 (IL-33Rα) are highly expressed in spinal cord tissue, and ST2 expression is markedly increased in the spinal cords of mice with EAE. Furthermore, ST2-deficient (ST2(-/-) ) mice developed exacerbated EAE compared with wild-type (WT) mice while WT, but not ST2(-/-) EAE mice treated with IL-33 developed significantly attenuated disease. IL-33-treated mice had reduced levels of IL-17 and IFN-γ but produced increased amounts of IL-5 and IL-13. Lymph node and splenic macrophages of IL-33-treated mice showed polarization toward an alternatively activated macrophage (M2) phenotype with significantly increased frequency of MR(+) PD-L2(+) cells. Importantly, adoptive transfer of these IL-33-treated macrophages attenuated EAE development. Our data therefore demonstrate that IL-33 plays a therapeutic role in autoimmune CNS disease by switching a predominantly pathogenic Th17/Th1 response to Th2 activity, and by polarization of anti-inflammatory M2 macrophages.

The pro-Th2 cytokine IL-33 is now emerging as an important Th1 cytokine-IFN-γ inducer in murine CD4(+) T cells that is essential for protective cell-mediated immunity against viral infection in mice. However, whether IL-33 can promote human Th1 cell differentiation and how IL-33 polarizes Th1 cells is less understood. We assessed the ability of IL-33 to induce Th1 cell differentiation and IFN-γ production in vitro and in vivo. We report here that IL-33 alone had no ability in Th1 cell polarization. However it potentiated IL-12-mediated Th1 cell differentiation and IFN-γ production in TCR-stimulated murine and human CD4(+) T cells in vitro and in vivo. IL-33 promoted Th1 cell development via MyD88 and synergized with IL-12 to enhance St2 and IL-12R expression in CD4(+) T cells. These data therefore provide a novel mechanism for Th1 cell differentiation and optimal induction of a Type 1 response. Thus, IL-33 is capable of inducing IL-12-dependent Th1 cell differentiation in human and mouse CD4(+) T cells.

Using TLR agonists in cancer treatment can have either beneficial or detrimental effects. Therefore, it is important to determine their effect on the tumor growth and understand the underlying mechanisms in animal tumor models. In this study, we report a general immunotherapeutic activity of a synthetic bacterial lipoprotein (BLP), a TLR1/TLR2 agonist, on established lung carcinoma, leukemia, and melanoma in mice. Systemic treatment of 3LL tumor-bearing mice with BLP, but not LPS, led to a dose-dependent tumor regression and a long-lasting protective response against tumor rechallenge. The BLP-mediated tumor remission was neither mediated by a direct tumoricidal activity nor by innate immune cells, because it lacked therapeutic effect in immunodeficient SCID mice. Instead, BLP treatment reduced the suppressive function of Foxp3+ regulatory T cells (Tregs) and enhanced the cytotoxicity of tumor-specific CTL in vitro and in vivo. Furthermore, adoptive cotransfer of BLP-pretreated but not untreated CTL and Tregs from wild-type but not from TLR2−/− mice was sufficient to restore antitumor immunity in SCID mice by reciprocally modulating Treg and CTL function. These results demonstrate that the TLR1/TLR2 agonist BLP may have a general tumor therapeutic property involving reciprocal downregulation of Treg and upregulation of CTL function. This property may play an important role in the development of novel antitumor strategies.

B1 B cells produce natural IgM and play a critical role in the early defense against bacterial and viral infection. The polyreactive IgM also contributes to the clearance of apoptotic products and plays an important role in autoimmune pathogenesis. However, the mechanism of activation and proliferation of B1 cells remains obscure. In this study, we report that IL-33, a new member of IL-1 family, activates B1 cells, which express the IL-33 receptor α, ST2. IL-33 markedly activated B1 cell proliferation and enhanced IgM, IL-5, and IL-13 production in vitro and in vivo in a ST2-dependent manner. The IL-33–activated B1 cell functions could be largely abolished by IL-5 neutralization and partially reduced by T cell or mast cell deficiency in vivo. ST2-deficient mice developed less severe oxazolone-induced contact sensitivity (CS) than did wild-type (WT) mice. Furthermore, IL-33 treatment significantly exacerbated CS in WT mice with enhanced B1 cell proliferation and IgM and IL-5 production. Moreover, IL-33–activated B1 cells from WT mice could adoptively transfer enhanced CS in ST2−/− mice challenged with IL-33. Thus, we demonstrate, to the best of our knowledge, a hitherto unrecognized mechanism of B1 cell activation and IL-33 function, and suggest that IL-33 may play an important role in delayed-type hypersensitivity.

Sepsis is a systemic inflammatory condition following bacterial infection with a high mortality rate and limited therapeutic options. Here we show that interleukin-33 (IL-33) reduces mortality in mice with experimental sepsis from cecal ligation and puncture (CLP). IL-33-treated mice developed increased neutrophil influx into the peritoneal cavity and more efficient bacterial clearance than untreated mice. IL-33 reduced the systemic but not the local proinflammatory response, and it did not induce a T helper type 1 (T(H)1) to T(H)2 shift. The chemokine receptor CXCR2 is crucial for recruitment of neutrophils from the circulation to the site of infection. Activation of Toll-like receptors (TLRs) in neutrophils downregulates CXCR2 expression and impairs neutrophil migration. We show here that IL-33 prevents the downregulation of CXCR2 and inhibition of chemotaxis induced by the activation of TLR4 in mouse and human neutrophils. Furthermore, we show that IL-33 reverses the TLR4-induced reduction of CXCR2 expression in neutrophils via the inhibition of expression of G protein-coupled receptor kinase-2 (GRK2), a serine-threonine protein kinase that induces internalization of chemokine receptors. Finally, we find that individuals who did not recover from sepsis had significantly more soluble ST2 (sST2, the decoy receptor of IL-33) than those who did recover. Together, our results indicate a previously undescribed mechanism of action of IL-33 and suggest a therapeutic potential of IL-33 in sepsis.

Objectives Interleukin 33 (IL-33) is a new member of the IL-1 family of cytokines which signals via its receptor, ST2 (IL-33R), and has an important role in Th2 and mast cell responses. This study shows that IL-33 orchestrates neutrophil migration in arthritis. Methods and results Methylated bovine serum albumin (mBSA) challenge in the knee joint of mBSA-immunised mice induced local neutrophil migration accompanied by increased IL-33R and IL-33 mRNA expression. Cell migration was inhibited by systemic and local treatments with soluble (s)IL-33R, an IL-33 decoy receptor, and was not evident in IL-33R-deficient mice. IL-33 injection also induced IL-33R-dependent neutrophil migration. Antigen- and IL-33-induced neutrophil migration in the joint was dependent on CXCL1, CCL3, tumour necrosis factor α (TNFα) and IL-1β synthesis. Synovial tissue, macrophages and activated neutrophils expressed IL-33R. IL-33 induces neutrophil migration by activating macrophages to produce chemokines and cytokines and by directly acting on neutrophils. Importantly, neutrophils from patients with rheumatoid arthritis successfully treated with anti-TNFα antibody (infliximab) expressed significantly lower levels of IL-33R than patients treated with methotrexate alone. Only neutrophils from patients treated with methotrexate alone or from normal donors stimulated with TNFα responded to IL-33 in chemotaxis. Conclusions These results suggest that suppression of IL-33R expression in neutrophils, preventing IL-33-induced neutrophil migration, may be an important mechanism of anti-TNFα therapy of inflammation.

Anaphylactic shock is characterized by elevated immunoglobulin-E (IgE) antibodies that signal via the high affinity Fcε receptor (FcεRI) to release inflammatory mediators. Here we report that the novel cytokine interleukin-33 (IL-33) potently induces anaphylactic shock in mice and is associated with the symptom in humans. IL-33 is a new member of the IL-1 family and the ligand for the orphan receptor ST2. In humans, the levels of IL-33 are substantially elevated in the blood of atopic patients during anaphylactic shock, and in inflamed skin tissue of atopic dermatitis patients. In murine experimental atopic models, IL-33 induced antigen-independent passive cutaneous and systemic anaphylaxis, in a T cell–independent, mast cell–dependent manner.In vitro, IL-33 directly induced degranulation, strong eicosanoid and cytokine production in IgE-sensitized mast cells. The molecular mechanisms triggering these responses include the activation of phospholipase D1 and sphingosine kinase1 to mediate calcium mobilization, Nuclear factor–κB activation, cytokine and eicosanoid secretion, and degranulation. This report therefore reveals a hitherto unrecognized pathophysiological role of IL-33 and suggests that IL-33 may be a potential therapeutic target for anaphylaxis, a disease of considerable unmet medical need.

LPS comprises a major PAMP and is a key target of the immune system during bacterial infection. While LPS can be recognised by innate immune cells via the TLR4 complex, it is unknown whether T lymphocytes, especially CD8(+) T cells are also capable of doing so. We report here that naive human CD8(+) T cells, after activation by TCR stimulation, express surface TLR4 and CD14. These activated CD8(+) T cells can then secrete high concentrations of IFN-gamma, granzyme and perforin in response to LPS. These effects can be specifically inhibited using siRNA for TLR4. Furthermore, LPS can synergize with IL-12 to polarize the CD8(+) T cells into cytotoxic T-cell 1 (Tc1) that produce IFN-gamma but not IL-4, with or without TCR activation. Moreover, CD8(+)CD45RO(+) memory T cells constitutively expressed TLR4 and markedly enhanced IFN-gamma production when stimulated with LPS. In contrast, activated murine CD8(+) T cells lack TLR4 and CD14 expression and fail to respond to LPS for proliferation and cytokine production. Thus, human but not murine CD8(+) T cells are able to directly recognise bacterial LPS via LPS receptor complex and TLR4 provides a novel signal for the activation of effector and memory human CD8(+) T cells.

Galectin-3 (Gal-3) is a member of the β-galactoside-binding lectin family and plays an important role in inflammation. However, the precise role of Gal-3 in autoimmune diseases remains obscure. We have investigated the functional role of Gal-3 in experimental autoimmune encephalomyelitis (EAE) following immunization with myelin oligodendrocyte glycoprotein (MOG)35–55 peptide. Gal-3 deficient (Gal-3−/−) mice developed significantly milder EAE and markedly reduced leukocyte infiltration in the CNS compared with similarly treated wild-type (WT) mice. Gal-3−/− mice also contained fewer monocytes and macrophages but more apoptotic cells in the CNS than did WT mice. Following Ag stimulation in vitro, lymph node cells from the immunized Gal-3−/− mice produced less IL-17 and IFN-γ than did those of the WT mice. In contrast, Gal-3−/− mice produced more serum IL-10, IL-5, and IL-13 and contained higher frequency of Foxp3+ regulatory T cells in the CNS than did the WT mice. Furthermore, bone marrow-derived dendritic cells from Gal-3−/− mice produced more IL-10 in response to LPS or bacterial lipoprotein than did WT marrow-derived dendritic cells. Moreover, Gal-3−/− dendritic cells induced Ag-specific T cells to produce more IL-10, IL-5, and IL-12, but less IL-17, than did WT dendritic cells. Taken together, our data demonstrate that Gal-3 plays an important disease-exacerbating role in EAE through its multifunctional roles in preventing cell apoptosis and increasing IL-17 and IFN-γ synthesis, but decreasing IL-10 production.

Detection of bacterial CpG-containing DNA (CpG DNA) by innate immune cells is dependent on toll-like receptor 9 (TLR9). Here we show that the expression of tlr9 mRNA was induced in mouse bone marrow-derived macrophages (BMMs) upon infection with the facultative Gram-negative intracellular bacterium Salmonella enterica serovar Typhimurium (S. typhimurium). Treatment of BMM with the inhibitory oligonucleotide (ODN) 2114, an antagonist of TLR9 signalling, enhanced intracellular S. typhimurium numbers approximately fivefold, whereas a control ODN (2310) had no significant effect. Surprisingly, 2114 also amplified S. typhimurium bacterial loads in TLR9-deficient BMM. Indeed, 2114 suppressed responses (nuclear factor-kappaB-dependent reporter gene expression and interleukin-12p40 secretion) to not only CpG DNA, but also the TLR2 ligand Pam(3)Cys, in BMM and RAW264 cells in a sequence-specific manner. Inhibitory ODNs, which have been proposed as therapeutic agents for the treatment of systemic lupus erythematosus because of their inhibitory effects on TLR9 signalling, may thus compromise the host response to bacterial pathogens through TLR9-independent mechanisms.

Galectin-3 is a β-galactoside-binding lectin that plays an important role in inflammatory diseases. It also interacts with the surface carbohydrates of many pathogens, including LPS. However, its role in infection is not fully understood. Data presented herein demonstrate for the first time that galectin-3 is a negative regulator of LPS-induced inflammation. Galectin-3 is constitutively produced by macrophages and directly binds to LPS. Galectin-3-deficient macrophages had markedly elevated LPS-induced signaling and inflammatory cytokine production compared with wild-type cells, which was specifically inhibited by the addition of recombinant galectin-3 protein. In contrast, blocking galectin-3 binding sites by using a neutralizing Ab or its ligand, β-lactose, enhanced LPS-induced inflammatory cytokine expression by wild-type macrophages. In vivo, mice lacking galectin-3 were more susceptible to LPS shock associated with excessive induction of inflammatory cytokines and NO production. However, these changes conferred greater resistance to Salmonella infection. Thus, galectin-3 is a previously unrecognized, naturally occurring, negative regulator of LPS function, which protects the host from endotoxin shock but, conversely, favors Salmonella survival.

IL-33 (IL-1F11) binds ST2 (IL-1R4), both of which are associated with optimal CD4+ Th2 polarization. Exogenous IL-33 drives induction of Th2-associated cytokines and associated pathological changes within the gut mucosa. Th2 polarization is also a prerequisite to expulsion of the intestinal-dwelling nematode Trichuris muris. In this study, we demonstrate that IL-33 mRNA is expressed early during parasite infection and susceptible mice can be induced to expel the parasite by a regime of exogenous IL-33 administration. IL-33 prevents an inappropriate parasite-specific Th1-polarized response and induces IL-4, IL-9, and IL-13. This redirection requires the presence of T cells and must occur at the initiation of the response to the pathogen. Interestingly, exogenous IL-33 also induced thymic stromal lymphopoietin mRNA within the infected caecum, an epithelial cell-restricted cytokine essential for the generation of Th2-driven parasite immunity. IL-33 also acts independently of T cells, altering intestinal pathology in chronically infected SCID mice, leading to an increased crypt length and intestinal epithelial cell proliferation, but reducing goblet cell hyperplasia. Thus, the ability of IL-33 to induce Th2 responses has functional relevance in the context of intestinal helminth infection, particularly during the initiation of the response. Copyright © 2008 by The American Association of Immunologists, Inc.

TLRs are primary sensors of both innate and adaptive immune systems, where they play a pivotal role in the response directed against structurally conserved components of pathogens. Synthetic bacterial lipopeptide Pam3CSK4 is a TLR2 agonist capable of modulating Th1 and Th2 responses. This study examines the therapeutic effect of Pam3CSK4 in established airway inflammation in a murine model of asthma. In mice previously sensitized and challenged with OVA, Pam3CSK4 given i.p. markedly reduced the total inflammatory cell infiltrate and eosinophilia in bronchoalveolar lavage fluid. Pam3CSK4 therapy was associated with a reduction in OVA-induced IL-4 and IL-5 secretion from thoracic lymph node culture, airways inflammation, bronchial hyperresponsiveness, and serum levels of IgE. Pam3CSK4 therapy was also associated with an increase in OVA-induced IFN-γ, IL-12, and IL-10 production. However, the anti-inflammatory effect of Pam3CSK4 was independent of IL-10 or TGF-β, but was critically dependent on IL-12, the production of which by dendritic cells was enhanced by Pam3CSK4 in vitro. Our results provide direct evidence that Pam3CSK4 could represent a novel therapeutic agent in allergic airways disease.

Toll is the founder of a group of pattern recognition receptors, which play a critical role in the innate immunity in Drosophila. At least 13 distinct Toll-like receptors (TLRs), recognising pathogen-associated molecular pattern (PAMPs), have now been identified in humans. Most investigations on TLRs have focused on cells of the innate system. We report here that naive human T cells expressed high levels of cell surface TLR2 after activation by anti-T cell receptor (TCR) antibody and interferon-alpha. Activated cells produced elevated levels of cytokines in response to the TLR2 ligand, bacterial lipopeptide (BLP). Furthermore, CD4(+)CD45RO(+) memory T cells from peripheral blood constitutively expressed TLR2 and produced IFNgamma in response to BLP. BLP also markedly enhanced the proliferation and IFNgamma production by CD45RO(+) T cells in the presence of IL-2 or IL-15. Thus, TLR2 serves as a co-stimulatory receptor for antigen-specific T cell development and participates in the maintenance of T cell memory. This suggests that pathogens, via their PAMPs, may contribute directly to the perpetuation and activation of long term T cell memory in both antigen dependent and independent manner.

We previously demonstrated that an attenuated strain of Salmonella enterica serovar Typhimurium, engineered to express IL-2 (strain GIDIL2), is cleared more rapidly than its parental, non-cytokine-expressing, strain (designated BRD509) from the reticuloendothelial system of susceptible BALB/c mice. This early clearance correlated with the induction of a strong innate immune response within a few hours of administration of GIDIL2 organisms. In the present study, we wished to assess the contribution of LPS recognition to GIDIL2-induced immune responses using Toll-like receptor 4 (TLR4) mutant C3H/HeJ mice. In contrast to LPS responder mice, both BRD509 and GIDIL2 strains persisted at higher levels in LPS non-responder animals. However, the GIDIL2 bacterial loads in the peritoneal cavity and spleen, recovered over a period of 21 days post infection, were consistently lower than the corresponding CFUs of the BRD509 strain. Direct evidence for the induction of innate immunity was shown by demonstrating increased NK cell cytotoxicity, NOS2 gene expression, and nitric oxide synthesis by peritoneal cells obtained as early as 2 h after infection with GIDIL2, but not BRD509, organisms. Unlike BALB/c mice, however, these responses failed to afford any protection against virulent challenge in C3H/HeJ mice. Taken together, our data demonstrate that despite the induction of innate immune responses by IL2-expressing organisms, this was not sufficient to induce protection in TLR4 mutant mice.

Cell locomotion is crucial to the induction of an effective immune response. We report here the chemoattraction of CD4+ T cells by IL-18, a member of the IL-1 cytokine family. Recombinant IL-18 increased the proportion of T cells in polarized morphology in vitro and stimulated their subsequent invasion into collagen gels in an IL-18 concentration gradient-dependent manner. Immunofluorescent microscopy studies determined that the major cell type responding to IL-18 was IL-18R+CD4+. Importantly, synovial CD4+ T cells from patients with rheumatoid arthritis responded to IL-18, adopting polarized morphology and gel invasion without further activation ex vivo, indicating the physiologic relevance of our observations. Finally, injection of rIL-18 into the footpad of DBA/1 mice led to local accumulation of inflammatory cells. These data therefore demonstrate for the first time lymphocyte chemoattractant properties of a member of the IL-1 cytokine family and its relevance in inflammatory diseases.

Regulatory T cells play a major role in modulating the immune response. However, most information on these cells centers on autoimmunity, and there is also considerable controversy on the functional characteristics of these cells. Here we provide direct in vitro and in vivo evidence that CD4+CD25+ regulatory T cells inhibit the differentiation and functions of both Th1 and Th2 cells. Importantly, CD4+CD25+ T cells suppressed the disease development of Leishmania major infection in SCID mice reconstituted with naive CD4+CD25− T cells. Furthermore, CD4+CD25+ T cells inhibited the development of colitis induced by both Th1 and Th2 cells in SCID mice. Our results therefore document that CD4+CD25+ regulatory T cells suppress both Th1 and Th2 cells and that these regulatory T cells have a profound therapeutic potential against diseases induced by both Th1 and Th2 cells in vivo.

T1/ST2L, an IL-1 receptor homologue, is selectively expressed on murine Th2 cells and specific anti-ST2L antibodies can profoundly modulate the Th1/Th2 balance in vivo. Naive CD4(+) T cells do not express ST2L but do so on activation with specific antigen in the presence of IL-4 or when stimulated with low doses of antigen in the absence of exogenously added IL-4. Similarly enhanced ST2L expression occurred after stimulation of Th2 cells with antigen or the mitogen ConA in the presence of APC. Restimulation of Th2 cells in the presence of IFN-gamma led to a decreased expression of ST2L to below basal levels. Conversely, Th2 cells cultured with IL-4 led to increased ST2L expression. The reduced expression of ST2L in response to high doses of antigen is also reversed by the neutralization of IFN-gamma. Using an ST2L promoter/luciferase reporter gene construct, we show that the distal but not proximal ST2L promoter is responsible for specific gene expression in Th2 cells. IL-4 enhances, whereas IFN-gamma suppresses ST2L expression via direct modulation of the distal promoter of the ST2L gene. These data provide a mechanistic explanation for the selective expression of ST2L on Th2 cells.

Background Asthma is thought to result from the generation of T helper type 2 (Th2) responses, leading to bronchial inflammation. IFN-λ1 (also known as IL-29) is a recently described member of the IFN-λ family and has been shown to decrease production of Th2 cytokines in vitro. However, the role and mechanism of IFN-λ1 in asthma remain unknown. Objectives The aim of this study was to clarify the importance of IFN-λ1 in allergen-induced airway hyperresponsiveness (AHR) and inflammation. Methods We used a murine model for ovalbumin (OVA)-induced asthma to examine the effect of intranasal delivery of recombinant adenovirus expressing human IFN-λ1 (Ad-hIFN-λ1) on AHR and allergic airway inflammation. Results Intranasal instillation of Ad-hIFN-λ1 before airway antigen challenge in OVA-immunized mice significantly decreased the severity of AHR and numbers of eosinophils and levels of IL-4, IL-5, and IL-13, but not IL-10 and IFN-γ; both in vivo, in the bronchoalveolar lavage fluid and in vitro, following stimulation of lymphocytes from spleens with OVA, compared with administration of a control virus (Ad-mock). Furthermore, Ad-hIFN-λ1 treatment inhibited serum IgE secretion and increased numbers of splenic CD4 + CD25 + FOXP3 + Treg cells. Histological studies showed that Ad-hIFN-λ1 attenuated OVA-induced lung tissue eosinophilia. Conclusions These results demonstrate that delivery of the Ad-hIFN-λ1 can mitigate allergic airway inflammation in experimental asthma. The potent immunoregulatory action of IFN-λ1 may offer a novel therapeutic approach to treat allergic asthma.