Monica Guma

NF-κB links innate immunity to the hypoxic response through transcriptional regulation of HIF-1α

The hypoxic response is an ancient stress response triggered by low ambient oxygen (O2) (ref. 1) and controlled by hypoxia-inducible transcription factor-1 (HIF-1), whose α subunit is rapidly degraded under normoxia but stabilized when O2-dependent prolyl hydroxylases (PHDs) that target its O2-dependent degradation domain are inhibited. Thus, the amount of HIF-1α, which controls genes involved in energy metabolism and angiogenesis, is regulated post-translationally. Another ancient stress response is the innate immune response, regulated by several transcription factors, among which NF-κB plays a central role. NF-κB activation is controlled by IκB kinases (IKK), mainly IKK-β, needed for phosphorylation-induced degradation of IκB inhibitors in response to infection and inflammation. IKK-β is modestly activated in hypoxic cell cultures when PHDs that attenuate its activation are inhibited. However, defining the relationship between NF-κB and HIF-1α has proven elusive. Using in vitro systems, it was reported that HIF-1α activates NF-κB, that NF-κB controls HIF-1α transcription and that HIF-1α activation may be concurrent with inhibition of NF-κB. Here we show, with the use of mice lacking IKK-β in different cell types, that NF-κB is a critical transcriptional activator of HIF-1α and that basal NF-κB activity is required for HIF-1α protein accumulation under hypoxia in cultured cells and in the liver and brain of hypoxic animals. IKK-β deficiency results in defective induction of HIF-1α target genes including vascular endothelial growth factor. IKK-β is also essential for HIF-1α accumulation in macrophages experiencing a bacterial infection. Hence, IKK-β is an important physiological contributor to the hypoxic response, linking it to innate immunity and inflammation.

Human Cytomegalovirus Infection Is Associated with Increased Proportions of NK Cells That Express the CD94/NKG2C Receptor in Aviremic HIV-1–Positive Patients

In healthy blood donors, serological positivity for human cytomegalovirus (HCMV) is associated with an increased proportion of NK cells bearing the CD94/NKG2C NK cell receptor (NKR). The expression of the activating CD94/NKG2C NKR and of the inhibitory CD94/NKG2A NKR was studied in a cohort of 45 aviremic human immunodeficiency virus type 1 (HIV-1)-positive patients receiving highly active antiretroviral therapy. The proportions of NKG2C+ NK cells were significantly increased in HIV-1-positive patients (mean +/- SD, 25.9% +/- 23.0%), compared with those in 31 healthy individuals (mean +/- SD, 16.1% +/- 20.7%). Yet, the association vanished when HCMV serological status was considered in a multivariate regression model. These results support the conclusion that changes in the NKR repertoire in HIV1-positive patients are related to a concomitant HCMV infection.

Caspase 1–independent activation of interleukin-1β in neutrophil-predominant inflammation

OBJECTIVE Interleukin-1beta (IL-1beta) is a key cytokine linked to the pathogenesis of acute arthritis. Caspase 1, neutrophil elastase, and chymase all process proIL-1beta to its biologically active form. This study was undertaken to examine the potential contributions of each of these proteases in experimental models of inflammatory arthritis. METHODS Caspase 1-deficient (Casp1-/-) and wild-type (WT) mice were tested for their response to arthritogenic K/BxN serum transfer for induction of arthritis or injection of monosodium urate monohydrate (MSU) crystals for induction of peritonitis. All mice were prophylactically treated with inhibitors of neutrophil elastase or chymase. Arthritic paws were tested for the presence of IL-1beta protein by enzyme-linked immunosorbent assay and Western blotting. Neutrophils and mast cells from WT and mutant mice were tested for their ability to secrete IL-1beta after in vitro stimulation, in the presence of protease inhibitors. RESULTS Casp1-/- and WT mice developed paw swelling to the same extent in the K/BxN serum transfer-induced arthritis model. MSU crystal injection into Casp1-/- mice also resulted in neutrophil influx and production of measurable peritoneal IL-1beta protein. Both of these responses were attenuated with neutrophil elastase inhibitors. K/BxN serum transfer-induced arthritis was also reduced by treatment with a chymase inhibitor. Casp1-/- neutrophils and mast cells, when exposed to MSU crystals, secreted similar amounts of IL-1beta protein upon in vitro stimulation with lipopolysaccharide, albeit at lower levels than that secreted by WT cells. Elastase and chymase inhibitors reduced the amount of IL-1beta released by these cells. CONCLUSION The production of IL-1beta by neutrophils and mast cells is not exclusively dependent on caspase 1, and other proteases can compensate for the loss of caspase 1 in vivo. These pathways might therefore compromise the caspase 1-targeted therapies in neutrophil-predominant arthritis.

Fibroblast-specific protein 1 identifies an inflammatory subpopulation of macrophages in the liver

Cirrhosis is the end result of chronic liver disease. Hepatic stellate cells (HSC) are believed to be the major source of collagen-producing myofibroblasts in cirrhotic livers. Portal fibroblasts, bone marrow-derived cells, and epithelial to mesenchymal transition (EMT) might also contribute to the myofibroblast population in damaged livers. Fibroblast-specific protein 1 (FSP1, also called S100A4) is considered a marker of fibroblasts in different organs undergoing tissue remodeling and is used to identify fibroblasts derived from EMT in several organs including the liver. The aim of this study was to characterize FSP1-positive cells in human and experimental liver disease. FSP1-positive cells were increased in human and mouse experimental liver injury including liver cancer. However, FSP1 was not expressed by HSC or type I collagen-producing fibroblasts. Likewise, FSP1-positive cells did not express classical myofibroblast markers, including αSMA and desmin, and were not myofibroblast precursors in injured livers as evaluated by genetic lineage tracing experiments. Surprisingly, FSP1-positive cells expressed F4/80 and other markers of the myeloid-monocytic lineage as evaluated by double immunofluorescence staining, cell fate tracking, flow cytometry, and transcriptional profiling. Similar results were obtained for bone marrow-derived and peritoneal macrophages. FSP1-positive cells were characterized by increased expression of COX2, osteopontin, inflammatory cytokines, and chemokines but reduced expression of MMP3 and TIMP3 compared with Kupffer cells/macrophages. These findings suggest that FSP1 is a marker of a specific subset of inflammatory macrophages in liver injury, fibrosis, and cancer.

Constitutive intestinal NF-κB does not trigger destructive inflammation unless accompanied by MAPK activation

Constitutive NF-κB activation in IECs induces inflammatory cytokines and chemokines in the lamina propria, but does not result in overt tissue damage unless acute inflammatory insults are present, causing TNF-dependent destruction and barrier disruption.

Expression and function of NKG2D in CD4+ T cells specific for human cytomegalovirus

The human NKG2D killer lectin‐like receptor (KLR) is coupled by the DAP10 adapter to phosphoinositide 3‐kinase (PI3 K) and specifically interacts with different stress‐inducible molecules (i.e. MICA, MICB, ULBP) displayed by some tumour and virus‐infected cells. This KLR is commonly expressed by human NK cells as well as TCRγδ+ and TCRαβ+CD8+ T lymphocytes, but it has been also detected in CD4+ T cells from rheumatoid arthritis and cancer patients. In the present study, we analysed NKG2D expression in human cytomegalovirus (HCMV)‐specific CD4+ T lymphocytes. In vitro stimulation of peripheral blood mononuclear cells (PBMC) from healthy seropositive individuals with HCMV promoted variable expansion of CD4+NKG2D+ T lymphocytes that coexpressed perforin. NKG2D was detected in CD28– and CD28dull subsets and was not systematically associated with the expression of other NK cell receptors (i.e. KIR, CD94/NKG2 and ILT2). Engagement of NKG2D with specific mAb synergized with TCR‐dependent activation of CD4+ T cells, triggering proliferation and cytokine production (i.e. IFN‐γ and TNF‐α). Altogether, the data support the notion that NKG2D functions as a prototypic costimulatory receptor in a subset of HCMV‐specific CD4+ T lymphocytes and thus may have a role in the response against infected HLA class II+ cells displaying NKG2D ligands.

Chronic epithelial NF-κB activation accelerates APC loss and intestinal tumor initiation through iNOS up-regulation

The role of NF-κB activation in tumor initiation has not been thoroughly investigated. We generated Ikkβ(EE)IEC transgenic mice expressing constitutively active IκB kinase β (IKKβ) in intestinal epithelial cells (IECs). Despite absence of destructive colonic inflammation, Ikkβ(EE)IEC mice developed intestinal tumors after a long latency. However, when crossed to mice with IEC-specific allelic deletion of the adenomatous polyposis coli (Apc) tumor suppressor locus, Ikkβ(EE)IEC mice exhibited more β-catenin+ early lesions and visible small intestinal and colonic tumors relative to Apc+/ΔIEC mice, and their survival was severely compromised. IEC of Ikkβ(EE)IEC mice expressed high amounts of inducible nitric oxide synthase (iNOS) and elevated DNA damage markers and contained more oxidative DNA lesions. Treatment of Ikkβ(EE)IEC/Apc+/ΔIEC mice with an iNOS inhibitor decreased DNA damage markers and reduced early β-catenin+ lesions and tumor load. The results suggest that persistent NF-κB activation in IEC may accelerate loss of heterozygocity by enhancing nitrosative DNA damage.

Differential effects of US2, US6 and US11 human cytomegalovirus proteins on HLA class Ia and HLA-E expression: impact on target susceptibility to NK cell subsets

We compared in an inducible expression system the individual effect of US2, US6 and US11 human cytomegalovirus (HCMV) proteins on HLA‐E and HLA class Ia surface expression, assessing in parallel their influence on target susceptibility to NK cell clones. To this end, the RPMI 8866 B lymphoma cell line (HLA‐A2, HLA‐A3, HLA‐B7, HLA‐Cw7, HLA‐ER, HLA‐EG) was stably cotransfected with the ecdysone receptor, together with the US sequences under the control of an ecdysone‐inducible promoter. Biosynthesis of viral proteins was turned on by incubating transfectants with Ponasterone A. US6 down‐regulated expression of all class I molecules, hampering target resistance to NK cell clones controlled by the CD94/NKG2A, KIR2DL2 and/or CD85j (ILT2 or LIR‐1) inhibitory receptors. By contrast, US11 reduced the surface levels of class Ia molecules but preserved HLA‐E; this rendered US11+ cells sensitive to NK clones under the control of KIR2DL2 and/or CD85j, while their resistance to CD94/NKG2A+KIR2DL2– effector cells was maintained. US2 preserved as well HLA‐E expression but selectively targeted class Ia molecules; in fact, HLA‐A and HLA‐C allotypes were down‐modulated whereas HLA‐B7 remained unaltered. US2+ targets became sensitive to KIR2DL2+ cells but remained resistant to CD94/NKG2A+CD85j+ NK clones. The differential effects of US proteins on HLA class Ia and HLA‐E likely reflect the evolutionary adaptation of HCMV to counteract NK‐mediated surveillance.

IgG4-related diseases

Immunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a fascinating condition recognised as a systemic disease in 2003 [1,2]. The first link between autoimmunity affecting the pancreas, elevated serum IgG4 concentrations and large numbers of IgG4-positive plasma cells in pancreatic tissue was described only 2 years earlier [3]. Since then, many diseases that have long been viewed organ-specific are now considered within the spectrum of IgG4-RD. Practically any organ can be affected, having in common a key pathological feature consisting in dense lymphocyte and plasma cell infiltrate rich in IgG4-positive plasma cells, storiform fibrosis and often an elevated serum IgG4 concentration. While good clinical response to steroid therapy is observed, immunosuppressive or B-cell depleting therapy can be required. It is important to distinguish the IgG4-RD from traditional organ-specific autoimmune disease to guide therapy.

NK Cell Receptors Involved in the Response to Human Cytomegalovirus Infection

Human cytomegalovirus (HCMV) infection is a paradigm of the complexity reached by host-pathogen interactions. To avoid recognition by cytotoxic T lymphocytes (CTL) HCMV inhibits the expression of HLA class I molecules. As a consequence, engagement of inhibitory killer immunoglobulin-like receptors (KIR), CD94/NKG2A, and CD85j (ILT2 or LIR-1) natural killer cell receptors (NKR) specific for HLA class I molecules is impaired, and infected cells become vulnerable to an NK cell response driven by activating receptors. In addition to the well-defined role of the NKG2D lectin-like molecule, the involvement of other triggering receptors (i.e., activating KIR, CD94/NKG2C, NKp46, NKp44, and NKp30) in the response to HCMV is being explored. To escape from NK cell-mediated surveillance, HCMV interferes with the expression of NKG2D ligands in infected cells. In addition, the virus may keep NK inhibitory receptors engaged preserving HLA class I molecules with a limited role in antigen presentation (i.e., HLA-E) or, alternatively, displaying class I surrogates. Despite considerable progress in the field, a number of issues regarding the involvement of NKR in the innate immune response to HCMV remain uncertain.