While it is known that monosodium urate (MSU) crystals cause the disease gout, the mechanism by which these crystals stimulate this inflammatory condition has not been clear. Here we find that the Toll/IL-1R (TIR) signal transduction adaptor myeloid differentiation primary response protein 88 (MyD88) is required for acute gouty inflammation. In contrast, other TIR adaptor molecules, TIRAP/Mal, TRIF, and TRAM, are not required for this process. The MyD88-dependent TLR1, -2, -4, -6, -7, -9, and -11 and IL-18 receptor (IL-18R) are not essential for MSU-induced inflammation. Moreover, MSU does not stimulate HEK cells expressing TLR1–11 to activate NF-κB. In contrast, mice deficient in the MyD88-dependent IL-1R showed reduced inflammatory responses, similar to those observed in MyD88-deficient mice. Similarly, mice treated with IL-1 neutralizing antibodies also showed reduced MSU-induced inflammation, demonstrating that IL-1 production and IL-1R activation play essential roles in MSU-triggered inflammation. IL-1R deficiency in bone marrow–derived cells did not affect the inflammatory response; however, it was required in non–bone marrow–derived cells. These results indicate that IL-1 is essential for the MSU-induced inflammatory response and that the requirement of MyD88 in this process is primarily through its function as an adaptor molecule in the IL-1R signaling pathway.
Chun-Jen Chen, Yan Shi, Arron Hearn, Kate Fitzgerald, Douglas Golenbock, George Reed, Shizuo Akira, Kenneth L. Rock
Nathalie Vergnolle, Laurie Cellars, Andrea Mencarelli, Giovanni Rizzo, Sunita Swaminathan, Paul Beck, Martin Steinhoff, Patricia Andrade-Gordon, Nigel W. Bunnett, Morley D. Hollenberg, John L. Wallace, Giuseppe Cirino, Stefano Fiorucci
Neutrophil NADPH oxidase plays a key role in host defense and in inflammation by releasing large amounts of superoxide and other ROSs. Proinflammatory cytokines such as GM-CSF and TNF-α prime ROS production by neutrophils through unknown mechanisms. Here we used peptide sequencing by tandem mass spectrometry to show that GM-CSF and TNF-α induce phosphorylation of Ser345 on p47phox, a cytosolic component of NADPH oxidase, in human neutrophils. As Ser345 is located in the MAPK consensus sequence, we tested the effects of MAPK inhibitors. Inhibitors of the ERK1/2 pathway abrogated GM-CSF–induced phosphorylation of Ser345, while p38 MAPK inhibitor abrogated TNF-α–induced phosphorylation of Ser345. Transfection of HL-60 cells with a mutated p47phox (S345A) inhibited GM-CSF– and TNF-α–induced priming of ROS production. This event was also inhibited in neutrophils by a cell-permeable peptide containing a TAT-p47phox-Ser345 sequence. Furthermore, ROS generation, p47phox-Ser345 phosphorylation, and ERK1/2 and p38 MAPK phosphorylation were increased in synovial neutrophils from rheumatoid arthritis (RA) patients, and TAT-Ser345 peptide inhibited ROS production by these primed neutrophils. This study therefore identifies convergent MAPK pathways on Ser345 that are involved in GM-CSF– and TNF-α–induced priming of neutrophils and are activated in RA. Inhibition of the point of convergence of these pathways might serve as a novel antiinflammatory strategy.
Pham My-Chan Dang, Allan Stensballe, Tarek Boussetta, Houssam Raad, Cedric Dewas, Yolande Kroviarski, Gilles Hayem, Ole N. Jensen, Marie-Anne Gougerot-Pocidalo, Jamel El-Benna
The IL-21 receptor (IL-21R) shows significant homology with the IL-4R, and CD4+ Th2 cells are an important source of IL-21. Here we examined whether the IL-21R regulates the development of Th2 responses in vivo. To do this, we infected IL-21R–/– mice with the Th2-inducing pathogens Schistosoma mansoni and Nippostrongylus brasiliensis and examined the influence of IL-21R deficiency on the development of Th2-dependent pathology. We showed that granulomatous inflammation and liver fibrosis were significantly reduced in S. mansoni–infected IL-21R–/– mice and in IL-21R+/+ mice treated with soluble IL-21R–Fc (sIL-21R–Fc). The impaired granulomatous response was also associated with a marked reduction in Th2 cytokine expression and function, as evidenced by the attenuated IL-4, IL-13, AMCase, Ym1, and FIZZ1 (also referred to as RELMα) responses in the tissues. A similarly impaired Th2 response was observed following N. brasiliensis infection. In vitro, IL-21 significantly augmented IL-4Rα and IL-13Rα1 expression in macrophages, resulting in increased FIZZ1 mRNA and arginase-1 activity following stimulation with IL-4 and IL-13. As such, these data identify the IL-21R as an important amplifier of alternative macrophage activation. Collectively, these results illustrate an essential function for the IL-21R in the development of pathogen-induced Th2 responses, which may have relevance in therapies for both inflammatory and chronic fibrotic diseases.
John Pesce, Mallika Kaviratne, Thirumalai R. Ramalingam, Robert W. Thompson, Joseph F. Urban, Allen W. Cheever, Deborah A. Young, Mary Collins, Michael J. Grusby, Thomas A. Wynn
CD4+CD25+ Tregs regulate immunity, but little is known about their own regulation. We now report that the human 60-kDa heat shock protein (HSP60) acts as a costimulator of human Tregs, both CD4+CD25int and CD4+CD25hi. Treatment of Tregs with HSP60, or its peptide p277, before anti-CD3 activation significantly enhanced the ability of relatively low concentrations of the Tregs to downregulate CD4+CD25– or CD8+ target T cells, detected as inhibition of target T cell proliferation and IFN-γ and TNF-α secretion. The enhancing effects of HSP60 costimulation on Tregs involved innate signaling via TLR2, led to activation of PKC, PI3K, and p38, and were further enhanced by inhibition of ERK. HSP60-treated Tregs suppressed target T cells both by cell-to-cell contact and by secretion of TGF-β and IL-10. In addition, the expression of ERK, NF-κB, and T-bet by downregulated target T cells was inhibited. Thus, HSP60, a self-molecule, can downregulate adaptive immune responses by upregulating Tregs innately through TLR2 signaling.
Alexandra Zanin-Zhorov, Liora Cahalon, Guy Tal, Raanan Margalit, Ofer Lider, Irun R. Cohen
The autoimmune disease immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) is caused by mutations in the forkhead box protein P3 (FOXP3) gene. In the mouse model of FOXP3 deficiency, the lack of CD4+CD25+ Tregs is responsible for lethal autoimmunity, indicating that FOXP3 is required for the differentiation of this Treg subset. We show that the number and phenotype of CD4+CD25+ T cells from IPEX patients are comparable to those of normal donors. CD4+CD25high T cells from IPEX patients who express FOXP3 protein suppressed the in vitro proliferation of effector T cells from normal donors, when activated by “weak” TCR stimuli. In contrast, the suppressive function of CD4+CD25high T cells from IPEX patients who do not express FOXP3 protein was profoundly impaired. Importantly, CD4+CD25high T cells from either FOXP3+ or FOXP3– IPEX patients showed altered suppression toward autologous effector T cells. Interestingly, IL-2 and IFN-γ production by PBMCs from IPEX patients was significantly decreased. These findings indicate that FOXP3 mutations in IPEX patients result in heterogeneous biological abnormalities, leading not necessarily to a lack of differentiation of CD4+CD25high Tregs but rather to a dysfunction in these cells and in effector T cells.
Rosa Bacchetta, Laura Passerini, Eleonora Gambineri, Minyue Dai, Sarah E. Allan, Lucia Perroni, Franca Dagna-Bricarelli, Claudia Sartirana, Susanne Matthes-Martin, Anita Lawitschka, Chiara Azzari, Steven F. Ziegler, Megan K. Levings, Maria Grazia Roncarolo
Histamine, signaling via the type 1 receptor (H1R), has been shown to suppress Th2 cytokine production by in vitro cultured T cells. We examined the role of H1R in allergic inflammation in vivo using a murine asthma model. Allergen-stimulated splenic T cells from sensitized H1R–/– mice exhibited enhanced Th2 cytokine production. Despite this Th2 bias, allergen-challenged H1R–/– mice exhibited diminished lung Th2 cytokine mRNA levels, airway inflammation, goblet cell metaplasia, and airway hyperresponsiveness (AHR). Restoration of pulmonary Th2 cytokines in H1R–/– mice by intranasal IL-4 or IL-13 restored inflammatory lung responses and AHR. Further investigation revealed that histamine acts as a T cell chemotactic factor and defective T cell trafficking was responsible for the absence of lung inflammation. Cultured T cells migrated in response to histamine in vitro, but this was ablated by blockade of H1R but not H2R. In vivo, allergen-specific WT but not H1R–/– CD4+ T cells were recruited to the lungs of naive recipients following inhaled allergen challenge. H1R–/– T cells failed to confer airway inflammation or AHR observed after transfer of WT T cells. Our data establish a role for histamine and H1R in promoting the migration of Th2 cells into sites of allergen exposure.
Paul J. Bryce, Clinton B. Mathias, Krista L. Harrison, Takeshi Watanabe, Raif S. Geha, Hans C. Oettgen
Uncontrolled mucosal immunity in the gastrointestinal tract of humans results in chronic inflammatory bowel disease (IBD), such as Crohn disease and ulcerative colitis. In early clinical trials as well as in animal models, IL-12 has been implicated as a major mediator of these diseases based on the ability of anti-p40 mAb treatment to reverse intestinal inflammation. The cytokine IL-23 shares the same p40 subunit with IL-12, and the anti-p40 mAbs used in human and mouse IBD studies neutralized the activities of both IL-12 and IL-23. IL-10–deficient mice spontaneously develop enterocolitis. To determine how IL-23 contributes to intestinal inflammation, we studied the disease susceptibility in the absence of either IL-23 or IL-12 in this model, as well as the ability of recombinant IL-23 to exacerbate IBD induced by T cell transfer. Our study shows that in these models, IL-23 is essential for manifestation of chronic intestinal inflammation, whereas IL-12 is not. A critical target of IL-23 is a unique subset of tissue-homing memory T cells, which are specifically activated by IL-23 to produce the proinflammatory mediators IL-17 and IL-6. This pathway may be responsible for chronic intestinal inflammation as well as other chronic autoimmune inflammatory diseases.
David Yen, Jeanne Cheung, Heleen Scheerens, Frédérique Poulet, Terrill McClanahan, Brent Mckenzie, Melanie A. Kleinschek, Alex Owyang, Jeanine Mattson, Wendy Blumenschein, Erin Murphy, Manjiri Sathe, Daniel J. Cua, Robert A. Kastelein, Donna Rennick
Lectin pathway activation of C3 is known to involve target recognition by mannan-binding lectin (MBL) or ficolins and generation of classical pathway C3 convertase via cleavage of C4 and C2 by MBL-associated serine protease 2 (MASP-2). We investigated C3 activation in C2-deficient human sera and in sera with other defined defects of complement to assess other mechanisms through which MBL might recruit complement. The capacity of serum to support C3 deposition was examined by ELISA using microtiter plates coated with O antigen–specific oligosaccharides derived from Salmonella typhimurium, S. thompson, and S. enteritidis corresponding to serogroups B, C, and D (BO, CO, and DO). MBL bound to CO, but not to BO and DO, and efficiently supported C3 deposition in the absence of C2, C4, or MASP-2. The existence of an MBL-dependent C2 bypass mechanism for alternative pathway–mediated C3 activation was clearly demonstrated using CO, solid-phase mannan, and E. coli LPS. MASP-1 might contribute, but was not required for C3 deposition in the model used. Independent of MBL, specific antibodies to CO supported C3 deposition through classical and alternative pathways. MBL-dependent C2 bypass activation could be particularly important in various inherited and acquired complement deficiency states.
Barbro Selander, Ulla Mårtensson, Andrej Weintraub, Eva Holmström, Misao Matsushita, Steffen Thiel, Jens C. Jensenius, Lennart Truedsson, Anders G. Sjöholm
When exposed to a pathogen, a naive CD4+ T cell is forced to make a cell fate decision that leads to a polarized population of Th1 IFN-γ– or Th2 IL-4– producing cells. Although IL-4 has traditionally been considered a factor that promotes Th2 cell differentiation, recent evidence has demonstrated that the site and timing of IL-4 expression in an immune response determines its ultimate effects on CD4+ T cell fate. Using a mast cell (MC) reconstitution model, we demonstrate that MC-derived IL-4 promoted Th1 responses in vivo. Furthermore, MCs from genetically disparate mouse strains varied in their potential for IL-4 expression. Independent of the activation mode, MCs from Th1-prone C57BL/6 mice exhibited a more robust Il4 response than did the Th2-prone strain Balb/c. The hierarchy of IL-4 expression potential was directly associated with the degree of basal chromatin accessibility at cis-regulatory elements conserved noncoding sequence–1 and VA enhancer within the Th2 locus. GATA1/2 and Ikaros, factors with opposing roles in chromatin remodeling, acted at these sites. We propose that GATA and Ikaros proteins coordinately fine-tune accessibility at the Il4 locus during development to variably regulate IL-4 expression. These events likely contribute to the genetically determined heterogeneity in Th1 responses that underlie susceptibility to many diseases.
Gregory D. Gregory, Shveta S. Raju, Susan Winandy, Melissa A. Brown