Analysis of Il36a induction by C/EBPß via a half-CRE•C/EBP element in murine macrophages in dependence of its CpG methylation level.

Interleukin-36α is a novel member of the IL-1 cytokine family that is highly expressed in epithelial tissues and several myeloid-derived cell types after induction. The transcription factor (TF) C/EBPß binds specifically to an essential half-CRE•C/EBP motif in the Il36a promoter to induce Il36a expression upon LPS stimulation. C/EBPs regulate gene expression by binding to recognition sequences that can contain 5'-cytosine-phosphate-guanine-3' dinucleotides (CpG), whose methylation can influence TF binding and gene expression. Herein we show that the half-CRE•C/EBP element in the Il36a promoter is differentially methylated in the murine RAW264.7 macrophage cell line and in primary murine macrophages. We demonstrate that C/EBPß binding to the half-CRE•C/EBP element in the Il36a promoter following LPS stimulation is insensitive to CpG methylation and that methylation of the CpG in the half-CRE•C/EBP element does not alter LPS-induced Il36a promoter activity which correlated with similar Il36a mRNA copy numbers and pro-IL-36α protein amount in both cell types. Taken together, our data indicate that C/EBPß binding to the half-CRE•C/EBP element and subsequent gene activation occurs independently of the CpG methylation status of the half-CRE•C/EBP motif and underlines the potential of C/EBPs to recognize methylated as well as unmethylated motifs.

C/EBPß is a transcriptional key regulator of IL-36α in murine macrophages.

Interleukin (IL)-36α - one of the novel members of the IL-1 family of cytokines - is a potent regulator of dendritic and T cells and plays an important role in inflammatory processes like experimental skin inflammation in mice and in mouse models for human psoriasis. Here, we demonstrate that C/EBPß, a transcription factor required for the selective expression of inflammatory genes, is a key activator of the Il36A gene in murine macrophages. RNAi-mediated suppression of C/EBPß expression in macrophages (C/EBPß(low) cells) significantly impaired Il36A gene induction following challenge with LPS. Despite the presence of five predicted C/EBP binding sites, luciferase reporter assays demonstrated that C/EBPß confers responsiveness to LPS primarily through a half-CRE•C/EBP element in the proximal Il36A promoter. Electrophoretic mobility shift assays showed that C/EBPß but not CREB proteins interact with this critical half-CRE•C/EBP element. In addition, overexpression of C/EBPß in C/EBPß(low) cells enhanced the expression of Il36A whereas CREB-1 had no effect. Finally, chromatin immunoprecipitation confirmed that C/EBPß but neither CREB-1, ATF-2 nor ATF4 is directly recruited to the proximal promoter region of the Il36A gene. Together, these findings demonstrate an essential role of C/EBPß in the regulation of the Il36A gene via the proximal half-CRE•C/EBP element in response to inflammatory stimuli.

Relevance of inducible nitric oxide synthase for immune control of Mycobacterium avium subspecies paratuberculosis infection in mice.

Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne's disease (JD), an incurable chronic intestinal bowel disease in ruminants. JD occurs worldwide and causes enormous economic burden in dairy industry. Research on JD pathobiology is hampered by its complexity which cannot completely be mimicked by small animal models. As a model the mouse allows dissecting some pathogenicity features of MAP. However, for unknown reasons MAP exhibits reduced growth in granulomas of infected mice compared to other Mycobacterium avium subspecies. Here, we characterized immune reactions of MAP-infected C57BL/6 mice. After infection, mice appeared fully immunocompetent. A strong antigen-specific T cell response was elicited indicated by IFNγ production of splenic T cells re-stimulated with MAP antigens. Function of splenic dendritic cells and proliferation of adoptively transferred antigen-specific CD4+ T cells was unaltered. Isolated splenic myeloid cells from infected mice revealed that MAP resides in CD11b+ macrophages. Importantly, sorted CD11b+CD11c- cells expressed high level of type 2 nitric oxide synthase (NOS2) but only low levels of pro- and anti-inflammatory cytokines. Correspondingly, MAP-infected MAC2 expressing myeloid cells in spleen and liver granuloma displayed strong expression of NOS2. In livers of infected Nos2-/-mice higher bacterial loads, more granuloma and larger areas of tissue damage were observed 5 weeks post infection compared to wild type mice. In vitro, MAP was sensitive to NO released by a NO-donor. Thus, a strong T cell response and concomitant NOS2/NO activity appears to control MAP infection, but allows development of chronicity and pathogen persistence. A similar mechanism might explain persistence of MAP in ruminants.

Presence of Infected Gr-1intCD11bhiCD11cint Monocytic Myeloid Derived Suppressor Cells Subverts T Cell Response and Is Associated With Impaired Dendritic Cell Function in Mycobacterium avium-Infected Mice.

Myeloid-derived suppressor cells (MDSC) are immature myeloid cells with immunomodulatory function. To study the mechanism by which MDSC affect antimicrobial immunity, we infected mice with two M. avium strains of differential virulence, highly virulent Mycobacterium avium subsp. avium strain 25291 (MAA) and low virulent Mycobacterium avium subsp. hominissuis strain 104 (MAH). Intraperitoneal infection with MAA, but not MAH, caused severe disease and massive splenic infiltration of monocytic MDSC (M-MDSC; Gr-1intCD11bhiCD11cint) expressing inducible NO synthase (Nos2) and bearing high numbers of mycobacteria. Depletion experiments demonstrated that M-MDSC were essential for disease progression. NO production by M-MDSC influenced antigen-uptake and processing by dendritic cells and proliferation of CD4+ T cells. M-MDSC were also induced in MAA-infected mice lacking Nos2. In these mice CD4+ T cell expansion and control of infection were restored. However, T cell inhibition was only partially relieved and arginase (Arg) 1-expressing M-MDSC were accumulated. Likewise, inhibition of Arg1 also partially rescued T cell proliferation. Thus, mycobacterial virulence results in the induction of M-MDSC that block the T cell response in a Nos2- and Arg1-dependent manner.

Upregulation of capsule enables Streptococcus pyogenes to evade immune recognition by antigen-specific antibodies directed to the G-related alpha2-macroglobulin-binding protein GRAB located on the bacterial surface.

One of the major problems associated with the development of a vaccine against Streptococcus pyogenes is the ability of this pathogen to escape recognition by antibodies directed against conserved surface-associated determinants and to establish infection in the setting of an acquired immune response. Identification of the mechanism(s) used by S. pyogenes to avoid recognition by antigen-specific antibodies and escape killing in blood was the focus of this study. We showed here that S. pyogenes was capable of surviving in human blood containing high levels of antibodies directed against the G-related alpha2-macroglobulin-binding protein GRAB, a highly conserved bacterial surface protein. S. pyogenes upregulated the hyaluronic acid capsule production during incubation with human blood, suggesting that the capsule may structurally minimize antibody access to protein GRAB. This hypothesis was confirmed by the ability of anti-GRAB antibodies to promote opsonophagocytosis of a capsule-deficient mutant of S. pyogenes but not of the encapsulated wild-type strain. Capsule upregulation and protection of S. pyogenes from opsonophagocytosis in the presence of anti-GRAB antibodies was also observed in a murine model of streptococcal infection. Thus, masking of surface immunogenic determinants by the hyaluronic acid capsule may constitute a novel mechanism of S. pyogenes for evasion of antigen-specific antibodies.

cGAS-STING-TBK1-IRF3/7 induced interferon-ß contributes to the clearing of non tuberculous mycobacterial infection in mice.

Type I interferons (IFN-I), such as IFN-α and IFN-ß are important messengers in the host response against bacterial infections. Knowledge about the role of IFN-I in infections by nontuberculous mycobacteria (NTM) is limited. Here we show that macrophages infected with pathogens of the Mycobacterium avium complex produced significantly lower amounts of IFN-ß than macrophages infected with the opportunistic pathogen M. smegmatis. To dissect the molecular mechanisms of this phenomenon, we focused on the obligate pathogen Mycobacterium avium ssp paratuberculosis (MAP) and the opportunistic M. smegmatis. Viability of both bacteria was required for induction of IFN-ß in macrophages. Both bacteria induced IFN-ß via the cGAS-STING-TBK1-IRF3/7-pathway of IFN-ß activation. Stronger phosphorylation of TBK1 and higher amounts of extracellular bacterial DNA in the macrophage cytosol were found in M. smegmatis infected macrophages than in MAP infected macrophages. After intraperitoneal infection of mice, a strong Ifnb induction by M. smegmatis correlated with clearance of the bacteria. In contrast, MAP only induced weak Ifnb expression which correlated with bacterial persistence and increased number of granulomas in the liver. In mice lacking the type I interferon receptor we observed improved survival of M. smegmatis while survival of MAP was similar to that in wildtype mice. On the other hand, treatment of MAP infected wildtype mice with the IFN-I inducer poly(I:C) or recombinant IFN-ß impaired the survival of MAP. This indicates an essential role of IFN-I in clearing infections by MAP and M. smegmatis. The expression level of IFN-I is decisive for transient versus persistent NTM infection.