Summary To explore whether bacterial secreted 4‐hydroxy‐2‐alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild‐type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor‐α and interleukin‐6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild‐type did not. The addition of 4‐hydroxy‐2‐heptylquinoline (HHQ) or 2‐heptyl‐3,4‐dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild‐type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor‐κB (NF‐κB) binding to its binding sites and the expression of NF‐κB target genes, and PQS delayed inhibitor of κB degradation, indicating that the effect of HHQ and PQS was mediated through the NF‐κB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.

The Th2 locus control region (LCR) has been shown to be important in efficient and coordinated cytokine gene regulation during Th2 cell differentiation. However, the molecular mechanism for this is poorly understood. To study the molecular mechanism of the Th2 LCR, we searched for proteins binding to it. We discovered that transcription factor YY1 bound to the LCR and the entire Th2 cytokine locus in a Th2-specific manner. Retroviral overexpression of YY1 induced Th2 cytokine expression. CD4-specific knockdown of YY1 in mice caused marked reduction in Th2 cytokine expression, repressed chromatin remodeling, decreased intrachromosomal interactions, and resistance in an animal model of asthma. YY1 physically associated with GATA-binding protein-3 (GATA3) and is required for GATA3 binding to the locus. YY1 bound to the regulatory elements in the locus before GATA3 binding. Thus, YY1 cooperates with GATA3 and is required for regulation of the Th2 cytokine locus and Th2 cell differentiation. T h2 cytokine genes il4, il13, and il5 are clustered in chromosome 5 in human and chromosome 11 in mouse. The expression of the Th2 cytokine genes are coordinately regulated during Th2 cell differentiation. Several different regulatory elements have been shown to play an important role in Th2 cytokine gene expression, including promoters of Th2 cytokine genes, enhancers [conserved noncoding sequence 1 (CNS1)/hypersensitive site 1-3 (HSS1-3), hypersensitive site V (HSV)/CNS2, and intronic enhancer (IE)/ HSII], a silencer (HSIV), and a locus control region (LCR) (1, 2). The functions of these regulatory elements in Th2 cytokine expression in Th2 cells in vivo have been thoroughly investigated in transgenic mice and knockout mice that have targeted deletion in the regulatory elements (3-10). Among these regulatory elements, Th2 LCR has been shown to coordinately regulate Th2 cytokine genes, to induce chromatin remodeling, and to be required for chromosomal interactions (1, 2). The Th2 LCR is located in the introns of the Rad50 gene and is composed of four DNase I hypersensitive sites, including RHS4, RHS5, RHS6, and RHS7 (11,12). In previous studies, we have shown that the Th2 LCR is essential for Th2 cytokine expression and chromosome remodeling in the Th2 cytokine locus and in the pathogenesis of allergic asthma (7,11,13).The entire Th2 cytokine locus undergoes chromatin remodeling during Th2 cell differentiation (1, 2, 14). DNase I hypersensitive sites, which reflect accessibility of chromatin, are developed at the specific regulatory regions during Th2 cell differentiation (15, 16). Histone 3 lysine 9 (H3K9)-acetylation and histone 3 lysine 4 (H3K4)-methylation increase at specific regulatory regions in the Th2 cytokine locus during Th2 cell differentiation (17)(18)(19)(20). Th2 cytokine locus also undergoes DNA demethylation during Th2 cell differentiation (11,(21)(22)(23)(24)(25)(26). Treatment with drugs that cause chromatin modification such as 5-azacytidine or Trichostatin A induces Th2 cytokine expression upon T-cell recep...

Previous studies have shown that Th2 cytokine genes on mouse chromosome 11 are coordinately regulated by the Th2 locus control region (LCR). To examine the in vivo function of Th2 LCR, we generated CD4-specific Th2 LCR-deficient (cLCR KO) mice using CreLoxP recombination. The number of CD4 T cells in the cLCR KO mouse was comparable to that in wild-type mice. The expression of Th2 cytokines was dramatically reduced in in vitro-stimulated naïve CD4 T cells. Deletion of the LCR led to a loss of general histone H3 acetylation and histone H3-K4 methylation, and demethylation of DNA in the Th2 cytokine locus. Upon ovalbumin challenge in the mouse model of allergic asthma, cLCR KO mice exhibited marked reduction in the recruitment of eosinophils and lymphocytes in the bronchoalveolar lavage fluid, serum IgE level, lung airway inflammation, mucus production in the airway walls, and airway hyperresponsiveness. These results directly demonstrate that the Th2 LCR is critically important in the regulation of Th2 cytokine genes, in chromatin remodeling of the Th2 cytokine locus, and in the pathogenesis of allergic asthma.chromatin remodeling | differentiation | locus control region C D4 T cells play an essential role in the activation and regulation of a variety of immune responses. Effector CD4 T cells are composed of several different subsets including Th1, Th2, and Th17 cells (1-5). The expression of subset-specific cytokines is critical for the differentiation and function of T helper cells (6, 7). The Th2 cytokine genes, il4, il5, and il13, are clustered on human chromosome 5 and mouse chromosome 11. The Th2 cytokine locus undergoes structural changes in its chromatin upon Th2 cell differentiation to accommodate the high level expression of Th2 cytokine genes. The changes include acquisition of DNase I hypersensitivity (8, 9), restriction enzyme accessibility (10), histone acetylation (11-16), histone methylation (17), and DNA demethylation (8,13,18,19). Chromatin remodeling and enhancement of transcription of a gene are regulated by trans-acting factors that bind to specific DNA regulatory elements. Many laboratories including ours have shown that the Th2 cytokine locus is regulated by a number of regulatory elements including enhancers [CNS-1/ HSS (9, 20-22), CNS-2/HSV (20, 23), IE/HSII (20)], a silencer (HSIV) (20,24), and a locus control region (LCR) (25).We have shown previously that the expression of Th2 cytokines is coordinately regulated by the Th2 LCR that is located in the 3′ region of the rad50 gene (25). The Th2 LCR is composed of four DNase I-hypersensitive sites, namely RHS4, RHS5, RHS6, and RHS7 (13, 26). We have shown that deletion of RHS7 causes marked reduction of Th2 cytokine genes under Th0 conditions and partial reduction under Th2 conditions (27), suggesting that RHS7 is important for Th2 cytokine expression. The Th2 LCR interacts with the promoters of Th2 cytokine genes through intrachromosomal associations (28). Deletion of RHS7 disrupts these (27), suggesting that RHS7 is critical for these int...

The transcription factors Bcl6 and Blimp1 have opposing roles in the development of the follicular helper T (TFH) cells: Bcl6 promotes and Blimp1 inhibits TFH cell differentiation. Similarly, Bcl6 activates, while Blimp1 represses, expression of the TFH cell marker PD-1. However, Bcl6 and Blimp1 repress each other’s expression, complicating the interpretation of the regulatory network. Here we sought to clarify the extent to which Bcl6 and Blimp1 independently control TFH cell differentiation by generating mice with T cell specific deletion of both Bcl6 and Blimp1 (double conditional KO [dcKO] mice). Our data indicate that Blimp1 does not control TFH cell differentiation independently of Bcl6. However, a population of T follicular regulatory (TFR) cells developed independently of Bcl6 in dcKO mice. We next analyzed regulation of IL-10 and PD-1, two genes controlled by both Bcl6 and Blimp1, and observed that Bcl6 regulates both genes independently of Blimp1. We found Bcl6 positively regulates PD-1 expression by inhibiting the ability of Tbet/Tbx21 to repress Pdcd1 transcription. Our data show a novel mechanism for positive control of gene expression by Bcl6, and illuminate how Bcl6 and Blimp1 control TFH cell differentiation.