CD4 helper T cells producing the pro-inflammatory cytokine IL17 (Th17) have been implicated in a number of inflammatory arthritides including the Spondyloarthritides. Th17 development is promoted by IL23. Ankylosing Spondylitis (AS), the commonest Spondyloarthritis, is genetically associated with both HLA-B27 (B27) and with IL23 receptor polymorphisms, however the link remains unexplained. We have previously shown that B27 can form heavy chain dimers (termed B272), which, unlike classical HLA-B27, bind the Killer-cell Immunoglobulin-like Receptor KIR3DL2. Here we show that B272-expressing antigen presenting cells stimulate the survival, proliferation and IL17 production of KIR3DL2+ CD4 T. KIR3DL2+ CD4 T cells are expanded and enriched for IL17 production in the blood and synovial fluid of patients with spondyloarthritis (SpA). Despite KIR3DL2+ cells comprising a mean of just 15% of CD4 T in the peripheral blood of SpA patients, this subset accounted for 70% of the observed increase in Th17 numbers in SpA subjects compared to controls. TCR-stimulated peripheral blood KIR3DL2+CD4 T cell lines from SpA patients secreted four fold more IL17 than KIR3DL2+ lines from controls or KIR3DL2-negative CD4 T. Strikingly, KIR3DL2+ CD4 T cells account for the majority of peripheral blood CD4 T cell IL23 receptor expression and produce more IL17 in the presence of IL23. Our findings link HLA-B27 with IL-17 production and suggest new therapeutic strategies in AS/SpA.

Tuberculosis is an infectious disease caused by Mycobacterium tuberculosis (Mtb). In the lungs, macrophages and neutrophils are the first immune cells that have contact with the infecting mycobacteria. Neutrophils are phagocytic cells that kill microorganisms through several mechanisms, which include the lytic enzymes and antimicrobial peptides that are found in their lysosomes, and the production of reactive oxygen species. Neutrophils also release extracellular vesicles (EVs) (100–1,000 nm in diameter) to the extracellular milieu; these EVs consist of a lipid bilayer surrounding a hydrophilic core and participate in intercellular communication. We previously demonstrated that human neutrophils infected in vitro with Mtb H37Rv release EVs (EV-TB), but the effect of these EVs on other cells relevant for the control of Mtb infection, such as macrophages, has not been completely analyzed. In this study, we characterized the EVs produced by non-stimulated human neutrophils (EV-NS), and the EVs produced by neutrophils stimulated with an activator (PMA), a peptide derived from bacterial proteins (fMLF) or Mtb, and observed that the four EVs differed in their size. Ligands for toll-like receptor (TLR) 2/6 were detected in EV-TB, and these EVs favored a modest increase in the expression of the co-stimulatory molecules CD80, a higher expression of CD86, and the production of higher amounts of TNF-α and IL-6, and of lower amounts of TGF-β, in autologous human macrophages, compared with the other EVs. EV-TB reduced the amount of intracellular Mtb in macrophages, and increased superoxide anion production in these cells. TLR2/6 ligation and superoxide anion production are known inducers of autophagy; accordingly, we found that EV-TB induced higher expression of the autophagy-related marker LC3-II in macrophages, and the co-localization of LC3-II with Mtb inside infected macrophages. The intracellular mycobacterial load increased when autophagy was inhibited with wortmannin in these cells. In conclusion, our results demonstrate that neutrophils produce different EVs in response to diverse activators, and that EV-TB activate macrophages and promote the clearance of intracellular Mtb through early superoxide anion production and autophagy induction, which is a novel role for neutrophil-derived EVs in the immune response to Mtb.

1Abstract The Human Leukocyte Antigen HLA-B27(B27) is strongly associated with the spondyloarthritides. B27 can be expressed at the cell surface of antigen presenting cells (APC) as both classical β2m-associated B27 and as B27 free heavy chain forms (FHC) including disulphide-bonded heavy chain homodimers (termed B272). B27 FHC forms but not classical B27 bind to KIR3DL2. HLA-A3 which is not associated with spondyloarthritis (SpA) is also a ligand for KIR3DL2. Here we show that B272 and B27 FHC bind more strongly to KIR3DL2 than other HLA-class I, including HLA-A3. B272 tetramers bound KIR3DL2 transfected cells more strongly than HLA-A3. KIR3DL2Fc bound to HLA-B27-transfected cells more strongly than to cells transfected with other HLA-class I. KIR3DL2Fc pulled down multimeric, dimeric and monomeric free heavy chains from HLA-B27 expressing cell lines. Binding to B272 and B27 FHC stimulated greater KIR3DL2 phosphorylation than HLA-A3. B272 and B27 FHC stimulated KIR3DL2CD3ε–transduced T cell IL-2 production to a greater extent than control HLA-class I. KIR3DL2 binding to B27 inhibited NK IFNγ secretion and promoted greater survival of KIR3DL2+CD4 T and NK cells than binding to other HLA-class I. KIR3DL2+ T cells from B27+SpA patients proliferated more in response to antigen presented by syngeneic APC than the same T cell subset from healthy and disease controls. Our results suggest that expansion of KIR3DL2-expressing leukocytes observed in B27+ SpA may be explained by the stronger interaction of KIR3DL2 with B27 FHC.

Objective. Spondylarthritides (SpA), including ankylosing spondylitis (AS), are common inflammatory rheumatic diseases that are strongly associated with positivity for the HLA class I allotype B27. HLA-B27 normally forms complexes with ␤ 2 -microglobulin (␤ 2 m) and peptide to form heterotrimers. However, an unusual characteristic of HLA-B27 is its ability to form ␤ 2 m-free heavy chain homodimers (HLA-B27 2 ), which, unlike classic HLA-B27, bind to killer cell immunoglobulinlike receptor 3DL2 (KIR-3DL2). Binding of HLA-B27 2 to KIR-3DL2-positive CD4؉ T and natural killer (NK) cells stimulates cell survival and modulates cytokine production. This study was undertaken to produce an antibody to HLA-B27 2 in order to confirm its expression in SpA and to inhibit its proinflammatory properties.Methods. We generated monoclonal antibodies by screening a human phage display library positively against B27 2 and negatively against B27 heterotrimers. Conclusion. These results demonstrate that antibody HD6 has potential for use in both the investigation and the treatment of AS and other B27-associated spondylarthritides.Positivity for HLA-B27 is strongly associated with ankylosing spondylitis (AS) and other spondylarthritides (SpA) (1-3). However, despite extensive investigation, understanding of the pathogenic role of HLA-B27 is limited (4). The canonical HLA-B27 heterotrimer structure comprises an HLA heavy chain that is noncovalently associated with a monomorphic ␤ 2 -microglobulin (␤ 2 m) light chain and a short peptide derived from self proteins, viruses, or bacteria. These heterotrimeric complexes form in the endoplasmic reticulum and egress to the cell surface, where they are recognized by CD8ϩ cytotoxic T cells through their T cell receptors (5). HLA-B27 can also form ␤ 2 m-free

BackgroundHeat shock protein 70 (Hsp70) is an intracellular chaperone protein with regulatory and cytoprotective functions. Hsp70 can also be found in the extracellular milieu, as a result of active secretion or passive release from damaged cells. The role of extracellular Hsp70 is not fully understood. Some studies report that it activates monocytes, macrophages and dendritic cells through innate immune receptors (such as Toll-like receptors, TLRs), while others report that Hsp70 is a negative regulator of the inflammatory response. In order to address this apparent inconsistency, in this study we evaluated the response of human monocytes to a highly purified recombinant Hsp70.MethodsHuman peripheral blood monocytes were stimulated with Hsp70, alone or in combination with TLR agonists. Cytokines were quantified in culture supernatants, their mRNAs were measured by RT-PCR, and the binding of transcription factors was evaluated by electrophoretic mobility shift assay (EMSA). Kruskal-Wallis test or one-way or two-way ANOVA were used to analyze the data.ResultsThe addition of Hsp70 to TLR-activated monocytes down-regulated TNF-α as well as IL-6 levels. This effect was independent of a physical interaction between Hsp70 and TLR agonists; instead it resulted of changes at the TNF-α gene expression level. The decrease in TNF-α expression correlated with the binding of HSF-1 (heat shock transcription factor 1, a transcription factor activated in response to Hsp70) and CHBF (constitutive HSE-binding factor) to the TNF-α gene promoter.ConclusionExtracellular Hsp70 negatively regulates the production of pro-inflammatory cytokines of monocytes exposed to TLR agonists and contributes to dampen the inflammatory response.

SummaryInflammation is necessary for survival, but it is also an important cause of human morbidity and mortality, as exemplified by sepsis. During inflammation, cells of the innate immune system are recruited and activated in response to infection, trauma or injury. These cells are activated through receptors, such as Toll-like receptors (TLRs), which recognize microbial ligands such as lipopolysaccharide (LPS). Triggering receptor expressed on myeloid cells (TREM)-1 amplifies the inflammatory response initiated by TLRs, and its expression on the surface of monocytes increases in the presence of TLR ligands. Here we have shown that in monocytes TREM-1 mRNA levels, measured by reverse transcription-polymerase chain reaction (RT-PCR), remained unchanged and TREM-1 protein levels, measured by flow cytometry, increased, indicating that LPS increases TREM-1 expression by a posttranscriptional mechanism. We also showed that TREM-1/Fc fusion protein decreased the ability of the sera of some patients with sepsis to activate monocytes, indicating that the TREM-1 ligand, whose identity is unknown, may be present in the sera of some of these patients. We describe a mechanism for the regulation of TREM-1 expression on monocytes and the possible presence of its ligand in serum; these findings help to explain the contribution of TREM-1 during systemic inflammation.

Possession of HLA-B27 (B27), strongly predisposes to the development of spondyloarthritis. B27 forms classical heterotrimeric complexes with beta-2-microglobulin (β2m) and peptide, and (β2m–free) free H chain (FHC) forms including B27 dimers (termed B272) at the cell surface. In this study we characterise the interaction of HLA-B27 with LILR, leukocyte Ig-like receptor (LILR)B1 and LILRB2 biophysically, biochemically and by FACS staining. LILRB1 bound to B27 heterotrimers with a KD of 5.3 ±1.5 μM but did not bind B27 FHC. LILRB2 bound to B272 and B27 FHC and B27 heterotrimers with KDs of 2.5, 2.6 and 22 ±6μM respectively. Domain exchange experiments showed that B272 bound to the two membrane distal Ig-like domains of LILRB2. In FACS staining experiments, B27 dimer protein and tetramers stained LILRB2 transfectants five times more strongly than B27 heterotrimers. Moreover, LILRB2Fc bound to dimeric and other B27 FHC forms on B27-expressing cell lines more strongly than other HLA-class I FHCs. B27 transfected cells expressing B27 dimers and FHC inhibited IL-2 production by LILRB2-expressing reporter cells to a greater extent than control HLA-class I transfectants. B27 heterotrimers complexed with the L6M variant of the GAG KK10 epitope bound with a similar affinity to complexes with the wild-type KK10 epitope (with KDs of 15.0±0.8 μM and 16.0±2.0 μM respectively). Disulfide-dependent B27 H chain dimers and multimers are stronger ligands for LILRB2 than HLA-class I heterotrimers and H chains. The stronger interaction of B27 dimers and FHC forms with LILRB2 compared with other HLA class I could play a role in spondyloarthritis pathogenesis.

Introduction Acute pancreatitis (AP) is usually a mild and selflimiting disease, but some patients develop a severe form that is associated with high mortality. In AP, local inflammation is followed first by the systemic inflammatory response syndrome and then by the compensatory anti-inflammatory response syndrome, which is defined by low human leukocyte antigen (HLA)-DR expression on monocytes, increased concentration of the anti-inflammatory cytokine IL-10, and decreased monocyte function. Our aim was to measure the expression of triggering receptor expressed on myeloid cells (TREM)-1 (a proposed marker of infection or inflammation) and HLA-DR on monocytes, and the serum concentrations of IL-6 (a proinflammatory cytokine) and IL-10 in patients with AP to determine whether these markers can identify patients at high risk of developing severe AP or infection.