NRROS negatively regulates reactive oxygen species during host defence and autoimmunity.

Reactive oxygen species (ROS) produced by phagocytes are essential for host defence against bacterial and fungal infections. Individuals with defective ROS production machinery develop chronic granulomatous disease. Conversely, excessive ROS can cause collateral tissue damage during inflammatory processes and therefore needs to be tightly regulated. Here we describe a protein, we termed negative regulator of ROS (NRROS), which limits ROS generation by phagocytes during inflammatory responses. NRROS expression in phagocytes can be repressed by inflammatory signals. NRROS-deficient phagocytes produce increased ROS upon inflammatory challenges, and mice lacking NRROS in their phagocytes show enhanced bactericidal activity against Escherichia coli and Listeria monocytogenes. Conversely, these mice develop severe experimental autoimmune encephalomyelitis owing to oxidative tissue damage in the central nervous system. Mechanistically, NRROS is localized to the endoplasmic reticulum, where it directly interacts with nascent NOX2 (also known as gp91(phox) and encoded by Cybb) monomer, one of the membrane-bound subunits of the NADPH oxidase complex, and facilitates the degradation of NOX2 through the endoplasmic-reticulum-associated degradation pathway. Thus, NRROS provides a hitherto undefined mechanism for regulating ROS production--one that enables phagocytes to produce higher amounts of ROS, if required to control invading pathogens, while minimizing unwanted collateral tissue damage.

Signaling via the IL-20 receptor inhibits cutaneous production of IL-1ß and IL-17A to promote infection with methicillin-resistant Staphylococcus aureus.

Staphylococcus aureus causes most infections of human skin and soft tissue and is a major infectious cause of mortality. Host defense mechanisms against S. aureus are incompletely understood. Interleukin 19 (IL-19), IL-20 and IL-24 signal through type I and type II IL-20 receptors and are associated with inflammatory skin diseases such as psoriasis and atopic dermatitis. We found here that those cytokines promoted cutaneous infection with S. aureus in mice by downregulating IL-1ß- and IL-17A-dependent pathways. We noted similar effects of those cytokines in human keratinocytes after exposure to S. aureus, and antibody blockade of the IL-20 receptor improved outcomes in infected mice. Our findings identify an immunosuppressive role for IL-19, IL-20 and IL-24 during infection that could be therapeutically targeted to alter susceptibility to infection.

Preserving immunogenicity of lethally irradiated viral and bacterial vaccine epitopes using a radio- protective Mn2+-Peptide complex from Deinococcus.

Although pathogen inactivation by γ-radiation is an attractive approach for whole-organism vaccine development, radiation doses required to ensure sterility also destroy immunogenic protein epitopes needed to mount protective immune responses. We demonstrate the use of a reconstituted manganous peptide complex from the radiation-resistant bacterium Deinococcus radiodurans to protect protein epitopes from radiation-induced damage and uncouple it from genome damage and organism killing. The Mn(2+) complex preserved antigenic structures in aqueous preparations of bacteriophage lambda, Venezuelan equine encephalitis virus, and Staphylococcus aureus during supralethal irradiation (25-40 kGy). An irradiated vaccine elicited both antibody and Th17 responses, and induced B and T cell-dependent protection against methicillin-resistant S. aureus (MRSA) in mice. Structural integrity of viruses and bacteria are shown to be preserved at radiation doses far above those which abolish infectivity. This approach could expedite vaccine production for emerging and established pathogens for which no protective vaccines exist.

Prostaglandin E2 suppresses antifungal immunity by inhibiting interferon regulatory factor 4 function and interleukin-17 expression in T cells.

T helper 17 (Th17) cells play an important role in mucosal host defense through production of the signature cytokines IL-17 and IL-22. Prostaglandin E2 (PGE2) has been shown to enhance IL-17 production by mature Th17 cells. However, when present during Th17 cell differentiation, we found that PGE2 inhibited the transcription factor IRF4 and suppressed production of IL-17 but not IL-22. We show that IRF4 was required for IL-17 expression but inhibited IL-22 expression, highlighting the potential for discordant regulation of these two cytokines in Th17 cells. The pathogenic fungus Cryptococcus neoformans produces PGE2, and we found that it uses PGE2- and IRF4-dependent mechanisms to specifically inhibit induction of IL-17 during Th17 cell differentiation. Blockade of host PGE2 during infection led to increased IL-17 production from CD4(+) T cells and increased survival of mice. These findings suggest that host- or pathogen-derived PGE2 can act directly on Th17 cells during differentiation to inhibit IL-17-dependent antimicrobial responses.

IL-22 bridges the lymphotoxin pathway with the maintenance of colonic lymphoid structures during infection with Citrobacter rodentium.

Colonic patches (CLPs) and isolated lymphoid follicles (ILFs) are two main lymphoid structures in the colon. Lymphoid tissue-inducer cells (LTi cells) are indispensable for the development of ILFs. LTi cells also produce interleukin 17 (IL-17) and IL-22, signature cytokines secreted by IL-17-producing helper T cells. Here we report that IL-22 acted downstream of the lymphotoxin pathway and regulated the organization and maintenance of mature CLPs and ILFs in the colon during infection with Citrobacter rodentium. Lymphotoxin (LTα(1)ß(2)) regulated the production of IL-22 during infection with C. rodentium, but the lymphotoxin-like protein LIGHT did not. IL-22 signaling was sufficient to restore the organization of CLPs and ILFs and host defense against infection with C. rodentium in mice lacking lymphotoxin signals, which suggests that IL-22 connects the lymphotoxin pathway to mucosal epithelial defense mechanisms.

Regulation and functions of the IL-10 family of cytokines in inflammation and disease.

The IL-10 family of cytokines consists of nine members: IL-10, IL-19, IL-20, IL-22, IL-24, IL-26, and the more distantly related IL-28A, IL-28B, and IL-29. Evolutionarily, IL-10 family cytokines emerged before the adaptive immune response. These cytokines elicit diverse host defense mechanisms, especially from epithelial cells, during various infections. IL-10 family cytokines are essential for maintaining the integrity and homeostasis of tissue epithelial layers. Members of this family can promote innate immune responses from tissue epithelia to limit the damage caused by viral and bacterial infections. These cytokines can also facilitate the tissue-healing process in injuries caused by infection or inflammation. Finally, IL-10 itself can repress proinflammatory responses and limit unnecessary tissue disruptions caused by inflammation. Thus, IL-10 family cytokines have indispensable functions in many infectious and inflammatory diseases.

Mucosal adjuvant activity of cholera toxin requires Th17 cells and protects against inhalation anthrax.

Cholera toxin (CT) elicits a mucosal immune response in mice when used as a vaccine adjuvant. The mechanisms by which CT exerts its adjuvant effects are incompletely understood. We show that protection against inhalation anthrax by an irradiated spore vaccine depends on CT-mediated induction of IL-17-producing CD4 Th17 cells. Furthermore, IL-17 is involved in the induction of serum and mucosal antibody responses by CT. Th17 cells induced by CT have a unique cytokine profile compared with those induced by IL-6 and TGF-beta, and their induction by CT requires cAMP-dependent secretion of IL-1beta and beta-calcitonin gene-related peptide by dendritic cells. These findings demonstrate that Th17 cells mediate mucosal adjuvant effects of CT and identify previously unexplored pathways involved in Th17 induction that could be targeted for development of unique mucosal adjuvants.

Phosphatidylserine receptor Tim-4 is essential for the maintenance of the homeostatic state of resident peritoneal macrophages.

Tim-4 is a phosphatidylserine (PS) receptor that is expressed on various macrophage subsets. It mediates phagocytosis of apoptotic cells by peritoneal macrophages. The in vivo functions of Tim-4 in phagocytosis and immune responses, however, are still unclear. In this study, we show that Tim-4 quickly forms punctate caps on contact with apoptotic cells, in contrast to its normal diffused expression on the surface of phagocytes. Despite its expression in marginal zone and tingible body macrophages, Tim-4 deficiency only minimally affects outcomes of several acute immune challenges, including the trapping of apoptotic cells in the marginal zone, the clearance apoptotic cells by tingible body macrophages, and the formation of germinal centers and elicitation of antibody responses against sheep red blood cells (SRBCs). In addition, Tim-4(-/-) resident peritoneal macrophages (rPMs) phagocytose necrotic cells and other opsonized targets normally. However, their ability to bind and engulf apoptotic cells is significantly compromised both in vitro and in vivo. Most importantly, Tim-4 deficiency results in increased cellularity in the peritoneum. Resting rPMs produce higher TNF-alpha in culture. Their response to LPS, on the contrary, is dampened. Our data support an indispensible role of Tim-4 in maintaining the homeostasis of rPMs.

Interleukin-22 mediates early host defense against attaching and effacing bacterial pathogens.

Infections by attaching and effacing (A/E) bacterial pathogens, such as Escherichia coli O157:H7, pose a serious threat to public health. Using a mouse A/E pathogen, Citrobacter rodentium, we show that interleukin-22 (IL-22) has a crucial role in the early phase of host defense against C. rodentium. Infection of IL-22 knockout mice results in increased intestinal epithelial damage, systemic bacterial burden and mortality. We also find that IL-23 is required for the early induction of IL-22 during C. rodentium infection, and adaptive immunity is not essential for the protective role of IL-22 in this model. Instead, IL-22 is required for the direct induction of the Reg family of antimicrobial proteins, including RegIIIbeta and RegIIIgamma, in colonic epithelial cells. Exogenous mouse or human RegIIIgamma substantially improves survival of IL-22 knockout mice after C. rodentium infection. Together, our data identify a new innate immune function for IL-22 in regulating early defense mechanisms against A/E bacterial pathogens.

The effects of IL-20 subfamily cytokines on reconstituted human epidermis suggest potential roles in cutaneous innate defense and pathogenic adaptive immunity in psoriasis.

IL-19, IL-20, IL-22, IL-24, and IL-26 are members of the IL-10 family of cytokines that have been shown to be up-regulated in psoriatic skin. Contrary to IL-10, these cytokines signal using receptor complex R1 subunits that are preferentially expressed on cells of epithelial origin; thus, we henceforth refer to them as the IL-20 subfamily cytokines. In this study, we show that primary human keratinocytes (KCs) express receptors for these cytokines and that IL-19, IL-20, IL-22, and IL-24 induce acanthosis in reconstituted human epidermis (RHE) in a dose-dependent manner. These cytokines also induce expression of the psoriasis-associated protein S100A7 and keratin 16 in RHE and cause persistent activation of Stat3 with nuclear localization. IL-22 had the most pronounced effects on KC proliferation and on the differentiation of KCs in RHE, inducing a decrease in the granular cell layer (hypogranulosis). Furthermore, gene expression analysis performed on cultured RHE treated with these cytokines showed that IL-19, IL-20, IL-22, and IL-24 regulate many of these same genes to variable degrees, inducing a gene expression profile consistent with inflammatory responses, wound healing re-epithelialization, and altered differentiation. Many of these genes have also been found to be up-regulated in psoriatic skin, including several chemokines, beta-defensins, S100 family proteins, and kallikreins. These results confirm that IL-20 subfamily cytokines are important regulators of epidermal KC biology with potentially pivotal roles in the immunopathology of psoriasis.

NTB-A, a new activating receptor in T cells that regulates autoimmune disease.

The CD28 co-stimulatory pathway is well established for T cell activation; however, results from CD28 -/- mice suggest the existence of additional co-stimulatory pathways. Here we report the further characterization of a new member of the CD2 superfamily, NTB-A, important in T cell co-stimulation. NTB-A is expressed on T cells, and its expression is up-regulated on activated cells. Triggering of NTB-A with monoclonal antibodies in the absence of CD28 signals leads to T cell proliferation and interferon-gamma secretion but not interleukin-4. Cross-linking of NTB-A also induces phosphorylation of NTB-A and the association of SAP (SLAM-associated protein), the protein absent in X-linked lymphoproliferative disease. T helper cells differentiated by cross-linking NTB-A and CD3 developed predominantly into Th1 cells not Th2 cells. In vivo blocking of NTB-A interactions with its ligands by using soluble NTB-A-Fc fusion protein inhibits B cell isotype switching to IgG2a and IgG3, commonly induced by Th1-type cytokines. Most important, treatment of mice with NTB-A-Fc delays the onset of antigen-induced experimental allergic encephalomyelitis in myelin basic protein-T cell receptor transgenic mice, suggesting a role in T cell-mediated autoimmune disease. Regulation of interferon-gamma secretion, and not interleukin-4 in vitro, as well as inhibition of Th1 cell-induced isotype switching and attenuation of experimental allergic encephalomyelitis indicate that NTB-A is important for Th1 responses. The observation that cross-linking of NTB-A induces T cell activation, expansion, and Th1-type cytokine production suggests NTB-A is a novel co-stimulatory receptor. The identification of NTB-A as a regulator of T cell response paves the way to provide novel therapeutic approaches for modulation of the immune response.