A single, oral dose of the TLR7 agonist JNJ-64794964 induces transcriptomic and phenotypic changes in peripheral immune cells in healthy adults.

BACKGROUND: Chronic hepatitis B (CHB) is responsible for major disease burden worldwide. However, the number of available therapies is limited; cure remains an elusive goal. JNJ-64794964 (JNJ-4964) is an oral toll-like receptor-7 (TLR7) agonist being evaluated for the treatment of CHB. Here, we investigated the capacity of JNJ-4964 to induce transcriptomic and immune cell changes in peripheral blood in healthy volunteers. METHODS: Peripheral blood was collected in the JNJ-4964 first-in-human phase 1 trial at multiple time points to assess transcriptomics and changes in frequency and phenotype of peripheral-blood mononuclear cells. Correlation of changes to JNJ-4964 exposure (Cmax) and changes in cytokine levels (C-X-C motif chemokine ligand 10 [CXCL10] and interferon alpha [IFN-α]) were evaluated. RESULTS: Fifty-nine genes, mainly interferon-stimulated genes, were up-regulated between 6 hours and 5 days after JNJ-4964 administration. JNJ-4964 increased frequencies of CD69, CD134, CD137, and/or CD253-expressing natural killer (NK) cells, indicative of NK cell activation. These changes correlated with Cmax, increase of CXCL10, and induction of IFN-α and were observed at IFN-α levels that are associated with no/acceptable flu-like adverse events. JNJ-4964 administration resulted in increased frequencies of CD86-expressing B cells, indicative of B-cell activation. These changes were predominantly observed at high IFN-α levels, which are associated with flu-like adverse events. CONCLUSIONS: JNJ-4964 administration led to changes in transcriptional profiles and immune cell activation phenotype, particularly for NK cells and B cells. Together, these changes could represent a set of biomarkers for the characterization of the immune response in CHB patients receiving TLR7 agonists.

Autoantibodies are associated with disease progression in idiopathic pulmonary fibrosis.

Several reports have highlighted a potential role of autoreactive B-cells and autoantibodies that correlates with increased disease severity in patients with idiopathic pulmonary fibrosis (IPF). Here we show that patients with IPF have an altered B-cell phenotype and that those subjects who have autoantibodies against the intermediate filament protein periplakin (PPL) have a significantly worse outcome in terms of progression-free survival. Using a mouse model of lung fibrosis, we demonstrate that introducing antibodies targeting the endogenous protein PPL (mimicking naturally occurring autoantibodies seen in patients) directly in the lung increases lung injury, inflammation, collagen and fibronectin expression through direct activation of follicular dendritic cells, which in turn activates and drives proliferation of fibroblasts. This fibrocyte population was also observed in fibrotic foci of patients with IPF and was increased in peripheral blood of IPF patients compared to aged-matched controls. This study reiterates the complex and heterogeneous nature of IPF, identifying new pathways that may prove suitable for therapeutic intervention.

Functional responses of resident human T cells in intact liver tissue.

The liver contains a rich mix of T cells, including activated T cells, tissue-resident memory T cells and cells undergoing apoptosis. When antigens are presented in this milieu the default result is functional tolerance. T cell tolerance in the liver could be constitutive, or it could be adaptive, in which case liver cells would become unresponsive after encountering antigen in the liver context. To test this model, we evaluated the potential of human liver T cells to respond to T cell receptor ligation in liver tissue slice cultures. These T cells contained an actively motile subset of CD4+ T cells marked by CCR7 and CD62L, and fully functional subsets of CD4+ and CD8+ T cells that synthesized effector cytokines but subsequently assumed an exhausted phenotype. These data favor the model that human liver T cells are not constitutively tolerant but undergo adaptive tolerance after activation.

Human Liver Macrophage Subsets Defined by CD32.

Human liver myeloid cells are imperfectly defined, but it is broadly agreed that cells of stellate appearance in situ, expressing the markers CD11b and CD68, are the liver's resident macrophages, classically termed Kupffer cells. Recent investigations using single cell RNA sequencing and unsupervised clustering algorithms suggest there are two populations of cells with the characteristics of tissue macrophages in human liver. We therefore analyzed dissociated human liver tissue using the markers CD11b and CD68 to define macrophage-like cells and found within this population two subsets that differ in their expression of multiple surface markers. These subsets were FACS-sorted based on CD32 expression, and gene expression analysis identified them with human liver myeloid cell subsets that were previously defined by two independent single cell RNA sequencing studies. Using qRT-PCR we found that the two subsets differed in the expression of genes associated with T cell activation and immunosuppression, suggesting distinct roles in T cell tolerance. In addition, one subset expressed two markers, CD1C and CD11c, more often seen on classical dendritic cells. Criteria used to distinguish macrophages from dendritic cells in other tissues may need to be revised in the human liver.

Moving toward endotypes in atopic dermatitis: Identification of patient clusters based on serum biomarker analysis.

BACKGROUND: Atopic dermatitis (AD) is a complex, chronic, inflammatory skin disease with a diverse clinical presentation. However, it is unclear whether this diversity exists at a biological level. OBJECTIVE: We sought to test the hypothesis that AD is heterogeneous at the biological level of individual inflammatory mediators. METHODS: Sera from 193 adult patients with moderate-to-severe AD (six area, six sign atopic dermatitis [SASSAD] score: geometric mean, 22.3 [95% CI, 21.3-23.3] and 39.1 [95% CI, 37.5-40.9], respectively) and 30 healthy control subjects without AD were analyzed for 147 serum mediators, total IgE levels, and 130 allergen-specific IgE levels. Population heterogeneity was assessed by using principal component analysis, followed by unsupervised k-means cluster analysis of the principal components. RESULTS: Patients with AD showed pronounced evidence of inflammation compared with healthy control subjects. Principal component analysis of data on sera from patients with AD revealed the presence of 4 potential clusters. Fifty-seven principal components described approximately 90% of the variance. Unsupervised k-means cluster analysis of the 57 largest principal components delivered 4 distinct clusters of patients with AD. Cluster 1 had high SASSAD scores and body surface areas with the highest levels of pulmonary and activation-regulated chemokine, tissue inhibitor of metalloproteinases 1, and soluble CD14. Cluster 2 had low SASSAD scores with the lowest levels of IFN-α, tissue inhibitor of metalloproteinases 1, and vascular endothelial growth factor. Cluster 3 had high SASSAD scores with the lowest levels of IFN-ß, IL-1, and epithelial cytokines. Cluster 4 had low SASSAD scores but the highest levels of the inflammatory markers IL-1, IL-4, IL-13, and thymic stromal lymphopoietin. CONCLUSION: AD is a heterogeneous disease both clinically and biologically. Four distinct clusters of patients with AD have been identified that could represent endotypes with unique biological mechanisms. Elucidation of these endotypes warrants further investigation and will require future intervention trials with specific agents, such as biologics.

Engineering the expression of an anti-interleukin-13 antibody through rational design and mutagenesis.

High levels of protein expression are key to the successful development and manufacture of a therapeutic antibody. Here, we describe two related antibodies, Ab001 and Ab008, where Ab001 shows a markedly lower level of expression relative to Ab008 when stably expressed in Chinese hamster ovary cells. We use single-gene expression vectors and structural analysis to show that the reduced titer is associated with the VL CDR2 of Ab001. We adopted two approaches to improve the expression of Ab001. First, we used mutagenesis to change single amino-acid residues in the Ab001 VL back to the equivalent Ab008 residues but this resulted in limited improvements in expression. In contrast when we used an in silico structure-based design approach to generate a set of five individual single-point variants in a discrete region of the VL, all exhibited significantly improved expression relative to Ab001. The most successful of these, D53N, exhibited a 25-fold increase in stable transfectants relative to Ab001. The functional potency of these VL-modified antibodies was unaffected. We expect that this in silico engineering strategy can be used to improve the expression of other antibodies and proteins.

Combinatorial Screening Identifies Novel Promiscuous Matrix Metalloproteinase Activities that Lead to Inhibition of the Therapeutic Target IL-13.

The practical realization of disease modulation by catalytic degradation of a therapeutic target protein suffers from the difficulty to identify candidate proteases, or to engineer their specificity. We identified 23 measurable, specific, and new protease activities using combinatorial screening of 27 human proteases against 24 therapeutic protein targets. We investigate the cleavage of monocyte chemoattractant protein 1, interleukin-6 (IL-6), and IL-13 by matrix metalloproteinases (MMPs) and serine proteases, and demonstrate that cleavage of IL-13 leads to potent inhibition of its biological activity in vitro. MMP-8 degraded human IL-13 most efficiently in vitro and ex vivo in human IL-13 transgenic mouse bronchoalveolar lavage. Hence, MMP-8 is a therapeutic protease lead against IL-13 for inflammatory conditions whereby reported genetic and genomics data suggest an involvement of MMP-8. This work describes the first exploitation of human enzyme promiscuity for therapeutic applications, and reveals both starting points for protease-based therapies and potential new regulatory networks in inflammatory disease.

Rituximab causes a polarization of B cells that augments its therapeutic function in NK-cell-mediated antibody-dependent cellular cytotoxicity.

Rituximab, which binds CD20 on B cells, is one of the best-characterized antibodies used in the treatment of B-cell malignancies and autoimmune diseases. Rituximab triggers natural killer (NK)-cell-mediated antibody-dependent cellular cytotoxicity (ADCC), but little is known about the spatial and temporal dynamics of cell-cell interactions during ADCC or what makes rituximab potent at triggering ADCC. Here, using laser scanning confocal microscopy, we found that rituximab caused CD20 to cap at the B-cell surface independent of antibody crosslinking or intercellular contact. Unexpectedly, other proteins, including intercellular adhesion molecule 1 and moesin, were selectively recruited to the cap of CD20 and the microtubule organizing center became polarized toward the cap. Importantly, the frequency at which NK cells would kill target cells via ADCC increased by 60% when target cells were polarized compared with when they were unpolarized. Polarized B cells were lysed more frequently still when initial contact with NK cells occurred at the place where CD20 was capped. This demonstrates that the site of contact between immune cells and target cells influences immune responses. Together, these data establish that rituximab causes a polarization of B cells and this augments its therapeutic function in triggering NK-cell-mediated ADCC.

The kinase PDK1 is essential for B-cell receptor mediated survival signaling.

Phosphoinositide-dependent kinase 1 (PDK1) plays an important role in integrating the T cell antigen receptor (TCR) and CD28 signals to achieve efficient NF-κB activation. PDK1 is also an important regulator of T cell development, mediating pre-TCR induced proliferation signals. However, the role of PDK1 in B cell antigen receptor (BCR) signaling and B cell development remains largely unknown. In this study we provide genetic evidence supporting the role of PDK1 in B cell survival. We found PDK1 is required for BCR mediated survival in resting B cells, likely through regulation of Foxo activation. PDK1-dependent signaling to NF-κB is not crucial to resting B cell viability. However, PDK1 is necessary for triggering NF-κB during B cell activation and is required for activated B cell survival. Together these studies demonstrate that PDK1 is essential for BCR-induced signal transduction to Foxo and NF-κB and is indispensable for both resting and activated B cell survival.

Nuclear factor-kappaB modulates regulatory T cell development by directly regulating expression of Foxp3 transcription factor.

Naturally derived regulatory T (Treg) cells are characterized by stable expression of the transcription factor Foxp3 and characteristic epigenetic imprinting at the Foxp3 gene locus. Here, we found that enhancing nuclear factor (NF)-kappaB activity via a constitutive active inhibitor of kappaB kinase beta (IKKbeta) transgene in T cells led to increased number of Foxp3(+) cells in the thymus and can rescue Foxp3 expression in thymocytes deficient in other pleiotropic signaling molecules. Enhancing the signal strength of the NF-kappaB pathway also induced Foxp3 expression in otherwise conventionally selected T cells. NF-kappaB directly promoted the transcription of Foxp3, and upon T cell receptor (TCR) stimulation, c-Rel, a NF-kappaB family member, bound to Foxp3 enhancer region, which is specifically demethylated in natural Treg cells. Hence, NF-kappaB signaling pathway is a key regulator of Foxp3 expression during natural Treg cell development.

Differential role of the transcription factor NF-kappaB in selection and survival of CD4+ and CD8+ thymocytes.

Inhibition of the transcription factor nuclear factor (NF)-kappaB activity leads to a reduction in numbers of CD8(+) single-positive (SP) thymocytes, suggesting a selective role for NF-kappaB in these cells. To further explore the role of NF-kappaB in SP thymocytes, we utilized transgenic models that allowed either inhibition or activation of NF-kappaB. We showed that activation of NF-kappaB played an important role in the selection of major histocompatibility complex (MHC) class I-restricted CD8(+) T cells. Surprisingly, NF-kappaB was not activated in positively selected CD4(+) thymocytes, and inhibition of NF-kappaB did not perturb positive or negative selection of CD4(+) cells. However, enforced activation of NF-kappaB via a constitutively active inhibitor of kappaB (IkappaB) kinase transgene led to a nearly complete deletion of CD4 cells by pushing positively selecting CD4(+) cells into negative selection. These studies therefore revealed a surprising difference of NF-kappaB activation in CD4(+) and CD8(+) thymocytes and suggested that NF-kappaB contributes to the establishment of thresholds of signaling that determine positive or negative selection of thymocytes.

Inhibition of NF-kappaB activation reduces the tissue effects of transgenic IL-13.

IL-13 is a major Th2 cytokine that is capable of inducing inflammation, excessive mucus production, airway hyperresponsiveness, alveolar remodeling, and fibrosis in the murine lung. Although IL-13 through its binding to IL-4Ralpha/IL-13Ralpha1 uses the canonical STAT6-signaling pathway to mediate these tissue responses, recent studies have demonstrated that other signaling pathways may also be involved. Previous studies from our laboratory demonstrated that IL-13 mediates its tissue effects by inducing a wide variety of downstream genes many of which are known to be regulated by NF-kappaB. As a result, we hypothesized that NF-kappaB activation plays a critical role in the pathogenesis of IL-13-induced tissue alterations. To test this hypothesis, we compared the effects of transgenic IL-13 in mice with normal and diminished levels of NF-kappaB activity. Three pharmacologic approaches were used to inhibit NF-kappaB including 1) PS1145, a small molecule inhibitor of IkappaBalpha kinase (IKK2), 2) antennapedia-linked NF-kappaB essential modulator-binding domain (NBD) peptide (wild-type NBD), and 3) an adenoviral construct expressing a dominant-negative version of IKK2. We also crossed IL-13-transgenic mice with mice with null mutations of p50 to generate mice that overproduced IL-13 in the presence and absence of this NF-kappaB component. These studies demonstrate that all these interventions reduced IL-13-induced tissue inflammation, fibrosis and alveolar remodeling. In addition, we show that both PS1145 and wild-type NBD inhibit lung inflammatory and structural cell apoptosis. PS1145 inhibits caspase activation and up-regulates inhibitor of apoptosis protein cellular-inhibitor of apoptosis protein 1 (c-IAP-1). Therefore, NF-kappaB is an attractive target for immunotherapy of IL-13-mediated diseases.

Investigation of mechanisms underlying the T-cell response to the hapten 2,4-dinitrochlorobenzene.

T-cell mediated contact sensitization by small molecular weight xenobiotics results in significant morbidity and absences from work. To be recognized by T-cells, xenobiotics must act as haptens, becoming protein-bound. At present, the requirement for processing and presentation of xenobiotics, the nature of the T-cell responses to them and the mechanisms that confer individual susceptibility in humans are unclear. We have investigated the T-cell response to the hapten 2,4-dinitrochlorobenzene (DNCB) which can sensitize all immunocompetent people. Fourteen healthy adults were sensitized with DNCB; 11 demonstrated positive T-cell responses to the chemical in vitro. Responding cells were of both CD4+ and CD8+ subsets, of Th1 and Tc1 phenotypes, producing high levels of IFN-gamma and low levels of IL-10. DNCB-specific T-cell clones were raised from 2 subjects, which in the presence of fixed and unfixed autologous Epstein-Barr virus transformed B cells as antigen-presenting-cells (APC), demonstrated that the chemical requires metabolic processing by the APC in order to initiate the T-cell response. Intracellular-reduced glutathione is consumed in detoxication of DNCB, leaving residual non-detoxified DNCB free to bind to proteins. The results suggest that DNCB forms multiple haptens with intracellular and extracellular proteins leading to Th1 and Tc1 responses in individuals exposed to this compound.

NF-kappa B activation in human breast cancer specimens and its role in cell proliferation and apoptosis.

Lack of molecular targets in estrogen receptor-negative (ER-negative) breast cancer is a major therapeutic hurdle. We studied NF-kappa B activation in human breast tumors and in carcinoma cell lines. Activated NF-kappa B was detected predominantly in ER-negative vs. ER-positive breast tumors and mostly in ER-negative and ErbB2-positive tumors (86%). These in vivo results demonstrate association of activated NF-kappa B with a subgroup of human breast tumors and are consistent with previously reported in vitro observations using similar classes of human breast cancer cell lines. Finding such an association suggested functional and biological significance. Immunofluorescence demonstrated increased nuclear p65, a component of the active NF-kappa B complex, in cytokeratin 19 (CK19)-positive epithelial cells of ER-negative/ErbB2-positive tumor samples. In contrast, nuclear NF-kappa B was detected mostly in stroma of ER-negative and ErbB2-negative tumors, suggesting a role of activated NF-kappa B in intercellular signaling between epithelial and stromal cells in this type of breast cancers. To elucidate roles of activated NF-kappa B, we used an ER-negative and ErbB2-positive human breast tumor cell line (SKBr3). The polypeptide heregulin beta1 stimulated, and herceptin, the anti-ErbB2 antibody, inhibited, NF-kappa B activation in SKBr3 cells. The NF-kappa B essential modulator (NEMO)-binding domain (NBD) peptide, an established selective inhibitor of I kappa B-kinase (IKK), blocked heregulin-mediated activation of NF-kappa B and cell proliferation, and simultaneously induced apoptosis only in proliferating and not resting cells. These results substantiate the hypothesis that certain breast cancer cells rely on NF-kappa B for aberrant cell proliferation and simultaneously avoid apoptosis, thus implicating activated NF-kappa B as a therapeutic target for distinctive subclasses of ER-negative breast cancers.

Characterization of two novel pyrogenic toxin superantigens made by an acute rheumatic fever clone of Streptococcus pyogenes associated with multiple disease outbreaks.

The pathogenesis of acute rheumatic fever (ARF) is poorly understood. We identified two contiguous bacteriophage genes, designated speL and speM, encoding novel inferred superantigens in the genome sequence of an ARF strain of serotype M18 group A streptococcus (GAS). speL and speM were located at the same genomic site in 33 serotype M18 isolates, and no nucleotide sequence diversity was observed in the 33 strains analyzed. Furthermore, the genes were absent in 13 non-M18 strains tested. These data indicate a recent acquisition event by a distinct clone of serotype M18 GAS. speL and speM were transcribed in vitro and upregulated in the exponential phase of growth. Purified SpeL and SpeM were pyrogenic and mitogenic for rabbit splenocytes and human peripheral blood mononuclear cells in picogram amounts. SpeL preferentially expanded human T cells expressing T-cell receptors Vbeta1, Vbeta5.1, and Vbeta23, and SpeM had specificity for Vbeta1 and Vbeta23 subsets, indicating that both proteins had superantigen activity. SpeL was lethal in two animal models of streptococcal toxic shock, and SpeM was lethal in one model. Serologic studies indicated that ARF patients were exposed to serotype M18 GAS, SpeL, and SpeM. The data demonstrate that SpeL and SpeM are pyrogenic toxin superantigens and suggest that they may participate in the host-pathogen interactions in some ARF patients.

Endogenous antimicrobial peptides and skin infections in atopic dermatitis.

BACKGROUND: The innate immune system of human skin contains antimicrobial peptides known as cathelicidins (LL-37) and beta-defensins. In normal skin these peptides are negligible, but they accumulate in skin affected by inflammatory diseases such as psoriasis. We compared the levels of expression of LL-37 and human beta-defensin 2 (HBD-2) in inflamed skin from patients with atopic dermatitis and from those with psoriasis. METHODS: The expression of LL-37 and HBD-2 protein in skin-biopsy specimens from patients with psoriasis, patients with atopic dermatitis, and normal subjects was determined by immunohistochemical analysis. The amount of antimicrobial peptides in extracts of skin samples was also analyzed by immunodot blot analysis (for LL-37) and Western blot analysis (for HBD-2). Quantitative, real-time reverse-transcriptase-polymerase-chain-reaction (RT-PCR) assays were used to confirm the relative expression of HBD-2 and LL-37 messenger RNA (mRNA) in the skin-biopsy specimens. These peptides were also tested for antimicrobial activity against Staphylococcus aureus with the use of a colony-forming assay. RESULTS: Immunohistochemical analysis confirmed the presence of abundant LL-37 and HBD-2 in the superficial epidermis of all patients with psoriasis. In comparison, immunostaining for these peptides was significantly decreased in acute and chronic lesions from patients with atopic dermatitis (P=0.006 and P=0.03, respectively). These results were confirmed by immunodot blot and Western blot analyses. Real-time RT-PCR showed significantly lower expression of HBD-2 mRNA and LL-37 mRNA in atopic lesions than in psoriatic lesions (P=0.009 and P=0.02, respectively). The combination of LL-37 and HBD-2 showed synergistic antimicrobial activity by effectively killing S. aureus. CONCLUSIONS: A deficiency in the expression of antimicrobial peptides may account for the susceptibility of patients with atopic dermatitis to skin infection with S. aureus.

Increased glucocorticoid receptor Beta expression converts mouse hybridoma cells to a corticosteroid-insensitive phenotype.

Glucocorticoid (GC) insensitivity is a challenging clinical problem associated with many chronic inflammatory disorders and life-threatening disease progression. The molecular basis of GC insensitivity, however, is unknown. Alternative splicing of the GC receptor (GCR) pre-mRNA generates a second GCR, termed GCRbeta, which does not bind GC but antagonizes the transactivating activity of the classic GCR, termed GCRalpha. GC-insensitive conditions have been associated with increased GCRbeta expression. Whether or not increased GCRbeta expression can contribute to GC insensitivity, however, remains controversial. To more precisely demonstrate the effect of GCRbeta on steroid responsiveness, we virally transduced GCRbeta cDNA into mouse DO-11.10 hybridoma cells, as mice are known to be deficient in the GCRbeta gene. We demonstrate that viral transduction of GCRbeta cDNA into mouse hybridoma cells to induce stable expression of GCRbeta results in GC insensitivity of these cells. Furthermore, in such cells GCRalpha is complexed with GCRbeta. Such heterodimer formation may account for the reduced effectiveness of GC action in cells overexpressing GCRbeta.