Stabilization of cytokine mRNAs in iNKT cells requires the serine-threonine kinase IRE1alpha.

Activated invariant natural killer T (iNKT) cells rapidly produce large amounts of cytokines, but how cytokine mRNAs are induced, stabilized and mobilized following iNKT activation is still unclear. Here we show that an endoplasmic reticulum stress sensor, inositol-requiring enzyme 1α (IRE1α), links key cellular processes required for iNKT cell effector functions in specific iNKT subsets, in which TCR-dependent activation of IRE1α is associated with downstream activation of p38 MAPK and the stabilization of preformed cytokine mRNAs. Importantly, genetic deletion of IRE1α in iNKT cells reduces cytokine production and protects mice from oxazolone colitis. We therefore propose that an IRE1α-dependent signaling cascade couples constitutive cytokine mRNA expression to the rapid induction of cytokine secretion and effector functions in activated iNKT cells.

A20 inhibition of STAT1 expression in myeloid cells: a novel endogenous regulatory mechanism preventing development of enthesitis.

OBJECTIVES: A20 is an important endogenous regulator of inflammation. Single nucleotide polymorphisms in A20 have been associated with various immune-mediated inflammatory diseases, and cell-specific deletion of A20 results in diverse inflammatory phenotypes. Our goal was to delineate the underlying mechanisms of joint inflammation in myeloid-specific A20-deficient mice (A20myelKO mice). METHODS: Inflammation in A20myelKO mice was assessed in a time-dependent manner. Western blot analysis and quantitative PCR analysis were performed on bone marrow-derived macrophages from A20myelKO and littermate control mice to study the effect of A20 on STAT1/STAT3 expression and STAT1/STAT3-dependent gene transcription in myeloid cells. The in vivo role of Janus kinase-Signal Transducer and Activator of Transcription (JAK-STAT) signalling in the development of enthesitis in A20myelKO mice was assessed following administration of a JAK inhibitor versus placebo control. RESULTS: Enthesitis was found to be an early inflammatory lesion in A20myelKO mice. A20 negatively modulated STAT1-dependent, but generally not STAT3-dependent gene transcription in myeloid cells by suppressing STAT1 but not STAT3 expression, both in unstimulated conditions and after interferon-γ or interleukin-6 stimulation. The increase in STAT1 gene transcription in the absence of A20 was shown to be JAK-STAT-dependent. Moreover, JAK inhibition in vivo resulted in significant reduction of enthesitis, both clinically and histopathologically. CONCLUSIONS: Our data reveal an important and novel interplay between myeloid cells and tissue resident cells at entheseal sites that is regulated by A20. In the absence of A20, STAT1 but not STAT3 expression is enhanced leading to STAT1-dependent inflammation. Therefore, A20 acts as a novel endogenous regulator of STAT1 that prevents onset of enthesitis.

NKT sublineage specification and survival requires the ubiquitin-modifying enzyme TNFAIP3/A20.

Natural killer T (NKT) cells are innate lymphocytes that differentiate into NKT1, NKT2, and NKT17 sublineages during development. However, the signaling events that control NKT sublineage specification and differentiation remain poorly understood. Here, we demonstrate that the ubiquitin-modifying enzyme TNFAIP3/A20, an upstream regulator of T cell receptor (TCR) signaling in T cells, is an essential cell-intrinsic regulator of NKT differentiation. A20 is differentially expressed during NKT cell development, regulates NKT cell maturation, and specifically controls the differentiation and survival of NKT1 and NKT2, but not NKT17, sublineages. Remaining A20-deficient NKT1 and NKT2 thymocytes are hyperactivated in vivo and secrete elevated levels of Th1 and Th2 cytokines after TCR ligation in vitro. Defective NKT development was restored by compound deficiency of MALT1, a key downstream component of TCR signaling in T cells. These findings therefore show that negative regulation of TCR signaling during NKT development controls the differentiation and survival of NKT1 and NKT2 cells.

How the microbiota shapes rheumatic diseases.

The human gut harbours a tremendously diverse and abundant microbial community that correlates with, and even modulates, many health-related processes. The mucosal interfaces are particularly active sites of microorganism-host interplay. Growing insight into the characteristic composition and functionality of the mucosal microbiota has revealed that the microbiota is involved in mucosal barrier integrity and immune function. This involvement affects proinflammatory and anti-inflammatory processes not only at the epithelial level, but also at remote sites such as the joints. Here, we review the role of the gut microbiota in shaping local and systemic immune responses and how disturbances in the host-microorganism interplay can potentially affect the development and progression of rheumatic diseases. Increasing our understanding of how to promote host-microorganism homeostasis could therefore reveal novel strategies for the prevention or alleviation of rheumatic disease.

Commensal microbiota influence systemic autoimmune responses.

Antinuclear antibodies are a hallmark feature of generalized autoimmune diseases, including systemic lupus erythematosus and systemic sclerosis. However, the processes underlying the loss of tolerance against nuclear self-constituents remain largely unresolved. Using mice deficient in lymphotoxin and Hox11, we report that approximately 25% of mice lacking secondary lymphoid organs spontaneously develop specific antinuclear antibodies. Interestingly, we find this phenotype is not caused by a defect in central tolerance. Rather, cell-specific deletion and in vivo lymphotoxin blockade link these systemic autoimmune responses to the formation of gut-associated lymphoid tissue in the neonatal period of life. We further demonstrate antinuclear antibody production is influenced by the presence of commensal gut flora, in particular increased colonization with segmented filamentous bacteria, and IL-17 receptor signaling. Together, these data indicate that neonatal colonization of gut microbiota influences generalized autoimmunity in adult life.

The thymic microenvironment differentially regulates development and trafficking of invariant NKT cell sublineages.

The regulatory role of the thymic microenvironment during trafficking and differentiation of the invariant NKT (iNKT) cell lineage remains poorly understood. In this study, we show that fractalkine receptor expression marks emigrating subpopulations of the NKT1, NKT2, and NKT17 sublineages in the thymus and peripheral organs of naive mice. Moreover, NKT1 sublineage cells can be subdivided into two subsets, namely NKT1(a) and NKT1(b), which exhibit distinct developmental and tissue-specific distribution profiles. More specifically, development and trafficking of the NKT1(a) subset are selectively dependent upon lymphotoxin (LT)α1ß2-LTß receptor-dependent differentiation of thymic stroma, whereas the NKT1(b), NKT2, and NKT17 sublineages are not. Furthermore, we identify a potential cellular source for LTα1ß2 during thymic organogenesis, marked by expression of IL-7Rα, which promotes differentiation of the NKT1(a) subset in a noncell-autonomous manner. Collectively, we propose a mechanism by which thymic differentiation and retention of the NKT1 sublineage are developmentally coupled to LTα1ß2-LTß receptor-dependent thymic organogenesis.

Invariant natural killer T cells are natural regulators of murine spondylarthritis.

OBJECTIVE: To investigate the role of invariant natural killer T (iNKT) cells in TNF(DeltaARE/+) mice, an animal model of spondylarthritis (SpA) with both gut and joint inflammation. METHODS: The frequency and activation of iNKT cells were analyzed on mononuclear cells from the lymph nodes and livers of mice, using flow cytometry with alpha-galactosylceramide/CD1d tetramers and quantitative polymerase chain reaction for the invariant V(alpha)14-J(alpha)18 rearrangement. Bone marrow-derived dendritic cells (DCs) were obtained by expansion of primary cells with granulocyte-macrophage colony-stimulating factor followed by coculture with iNKT cell hybridomas, and interleukin-2 release into the cocultures was then measured by enzyme-linked immunosorbent assay (ELISA). Cytokine levels were determined by ELISA or cytometric bead array analyses of freshly isolated DCs and iNKT cells in mixed cocultures. TNF(DeltaARE/+) mice were backcrossed onto J(alpha)18(-/-) and CD1d(-/-) mice, and disease onset was evaluated by clinical scoring, positron emission tomography, and histology. CD1d levels were analyzed on mononuclear cells in paired blood and synovial fluid samples from patients with SpA compared with healthy control subjects. RESULTS: In the absence of iNKT cells, symptoms of gut and joint inflammation in TNF(DeltaARE/+)mice were aggravated. Invariant NKT cells were activated during the course of the disease. This was linked to an enrichment of inflammatory DCs, characterized by high levels of CD1d, particularly at draining sites of inflammation. A similar increase in CD1d levels was observed on DCs from patients with SpA. Inflammatory DCs from TNF(DeltaARE/+) mice stimulated iNKT cells to produce immunomodulatory cytokines, in the absence of exogenous stimulation. Prolonged, continuous exposure, but not short-term exposure, to tumor necrosis factor (TNF) was found to be responsible for the enhanced DC-NKT cell crosstalk. CONCLUSION: This mode of iNKT cell activation represents a natural counterregulatory mechanism for the dampening of TNF-driven inflammation.

Invariant natural killer T cells in rheumatic disease: a joint dilemma.

Invariant natural killer T (iNKT) cells are an innate T-cell lineage known to recognize a range of endogenously derived and exogenously derived glycolipid antigens. Advances in our understanding of this T-cell subset have enabled researchers to investigate the immunomodulatory activity of iNKT cell ligands in experimental models of diseases such as cancer, allergy and chronic inflammatory joint disease. To a large extent, the ability of iNKT cells to regulate such disease models has been ascribed to their capacity to promote a polarized cytokine environment, which is understood to skew adaptive immune responses. In this Review, we discuss the current understanding of how iNKT-cell polarization is regulated and relate this basic theory to the proposed role for iNKT cells in models of rheumatologic disease.

Cutting edge: the chemokine receptor CXCR3 retains invariant NK T cells in the thymus.

The current model used to define T cell export from the thymus suggests that emigrating lymphocytes seed the peripheral organs as functionally mature cells. This model holds true for the majority of T cells exported from the thymus with the exception of invariant NK T (iNKT) cells. iNKT cells undergo lineage expansion after positive selection and acquire NK receptor expression once fully mature; yet, the majority of mature iNKT cells are retained in the thymus by an as of yet unidentified mechanism. In this study we demonstrate that mature iNKT cells are retained in the thymus by the chemokine receptor CXCR3. We propose that the expression of CXCR3 ligands in the thymic medullary epithelium promotes the chemotactic retention of mature iNKT thymocytes and prevents leakage of iNKT cells into the peripheral circulation.

Thymic emigration: sphingosine-1-phosphate receptor-1-dependent models and beyond.

The thymus is a primary lymphoid organ supporting the development of self-tolerant T cells. Key events in T-cell development in the thymus include lineage commitment, selection events, and thymic emigration. This review discusses the proposed role of sphingosine-1-phosphate and its receptors in the emigration of both conventional and unconventional T-cell subsets from the thymus, and the molecular machinery currently understood to regulate this process. Furthermore, we highlight a role for chemokines and actin-associated proteins in T-cell motility as recent data suggest that T-cell emigration is regulated by more than just a sphingosine-1-phosphate receptor-1-dependent chemotactic axis.

The induction of a type 1 immune response following a Trypanosoma brucei infection is MyD88 dependent.

The initial host response toward the extracellular parasite Trypanosoma brucei is characterized by the early release of inflammatory mediators associated with a type 1 immune response. In this study, we show that this inflammatory response is dependent on activation of the innate immune system mediated by the adaptor molecule MyD88. In the present study, MyD88-deficient macrophages are nonresponsive toward both soluble variant-specific surface glycoprotein (VSG), as well as membrane-bound VSG purified from T. brucei. Infection of MyD88-deficient mice with either clonal or nonclonal stocks of T. brucei resulted in elevated levels of parasitemia. This was accompanied by reduced plasma IFN-gamma and TNF levels during the initial stage of infection, followed by moderately lower VSG-specific IgG2a Ab titers during the chronic stages of infection. Analysis of several TLR-deficient mice revealed a partial requirement for TLR9 in the production of IFN-gamma and VSG-specific IgG2a Ab levels during T. brucei infections. These results implicate the mammalian TLR family and MyD88 signaling in the innate immune recognition of T. brucei.

Toll-like receptor 2-deficient mice succumb to Mycobacterium tuberculosis infection.

Recognition of Mycobacterium tuberculosis by the innate immune system is essential in the development of an adaptive immune response. Mycobacterial cell wall components activate macrophages through Toll-like receptor (TLR) 2, suggesting that this innate immune receptor plays a role in the host response to M. tuberculosis infection. After aerosol infection with either 100 or 500 live mycobacteria, TLR2-deficient mice display reduced bacterial clearance, a defective granulomatous response, and develop chronic pneumonia. Analysis of pulmonary immune responses in TLR2-deficient mice after 500 mycobacterial aerosol challenge showed increased levels of interferon-gamma, tumor necrosis factor-alpha, and interleukin-12p40 as well as increased numbers of CD4(+) and CD8(+) cells. Furthermore, TLR2-deficient mice mounted elevated Ag-specific type 1 T-cell responses that were not protective because all deficient mice succumb to infection within 5 months. Taken together, the data suggests that TLR2 may function as a regulator of inflammation, and in its absence an exaggerated immune inflammatory response develops.