Therapeutic TNF Inhibitors Exhibit Differential Levels of Efficacy in Accelerating Cutaneous Wound Healing.

Adalimumab but neither etanercept nor certolizumab-pegol has been reported to induce a wound-healing profile in vitro by regulating macrophage differentiation and matrix metalloproteinase expression, which may underlie the differences in efficacy between various TNF-α inhibitors in impaired wound healing in patients with hidradenitis suppurativa, a chronic inflammatory skin disease. To examine and compare the efficacy of various TNF inhibitors in cutaneous wound healing in vivo, a human TNF knock-in Leprdb/db mouse model was established to model the impaired cutaneous wound healing as seen in hidradenitis suppurativa. The vehicle group exhibited severe impairments in cutaneous wound healing. In contrast, adalimumab significantly accelerated healing, confirmed by both histologic assessment and a unique healing transcriptional profile. Moreover, adalimumab and infliximab showed similar levels of efficacy, but golimumab was less effective, along with etanercept and certolizumab-pegol. In line with histologic assessments, proteomics analyses from healing wounds exposed to various TNF inhibitors revealed distinct and differential wound-healing signatures that may underlie the differential efficacy of these inhibitors in accelerating cutaneous wound healing. Taken together, these data revealed that TNF inhibitors exhibited differential levels of efficacy in accelerating cutaneous wound healing in the impaired wound-healing model in vivo.

The Clinical Response of Upadacitinib and Risankizumab Is Associated With Reduced Inflammatory Bowel Disease Anti-TNF-α Inadequate Response Mechanisms.

BACKGROUND: Janus kinase (JAK) 1 inhibitor upadacitinib and IL-23 inhibitor risankizumab are efficacious in inflammatory bowel disease (IBD) patients who are antitumor necrosis factor (anti-TNF)-α inadequate responders (TNF-IRs). We aimed to understand the mechanisms mediating the response of upadacitinib and risankizumab. METHODS: Eight tissue transcriptomic data sets from IBD patients treated with anti-TNF-α therapies along with single-cell RNAseq data from ulcerative colitis were integrated to identify TNF-IR mechanisms. The RNAseq colon tissue data from clinical studies of TNF-IR Crohn's disease patients treated with upadacitinib or risankizumab were used to identify TNF-IR mechanisms that were favorably modified by upadacitinib and risankizumab. RESULTS: We found 7 TNF-IR upregulated modules related to innate/adaptive immune responses, interferon signaling, and tissue remodeling and 6 TNF-IR upregulated cell types related to inflammatory fibroblasts, postcapillary venules, inflammatory monocytes, macrophages, dendritic cells, and cycling B cells. Upadacitinib was associated with a significant decrease in the expression of most TNF-IR upregulated modules in JAK1 responders (JAK1-R); in contrast, there was no change in these modules among TNF-IR patients treated with a placebo or among JAK1 inadequate responders (JAK1-IR). In addition, 4 of the 6 TNF-IR upregulated cell types were significantly decreased after upadacitinib treatment in JAK1-R but not among subjects treated with a placebo or among JAK1-IR patients. We observed similar findings from colon biopsy samples from TNF-IR patients treated with risankizumab. CONCLUSIONS: Collectively, these data suggest that upadacitinib and risankizumab affect TNF-IR upregulated mechanisms, which may account for their clinical response among TNF-IR IBD patients.

We identified molecular and cellular mechanisms associated with and potentially mediating the response of upadacitinib and risankizumab for IBD patients that inadequately responded to anti-TNF-α treatment.

A population of proinflammatory T cells coexpresses αß and γδ T cell receptors in mice and humans.

T cells are classically recognized as distinct subsets that express αß or γδ TCRs. We identify a novel population of T cells that coexpress αß and γδ TCRs in mice and humans. These hybrid αß-γδ T cells arose in the murine fetal thymus by day 16 of ontogeny, underwent αß TCR-mediated positive selection into CD4+ or CD8+ thymocytes, and constituted up to 10% of TCRδ+ cells in lymphoid organs. They expressed high levels of IL-1R1 and IL-23R and secreted IFN-γ, IL-17, and GM-CSF in response to canonically restricted peptide antigens or stimulation with IL-1ß and IL-23. Hybrid αß-γδ T cells were transcriptomically distinct from conventional γδ T cells and displayed a hyperinflammatory phenotype enriched for chemokine receptors and homing molecules that facilitate migration to sites of inflammation. These proinflammatory T cells promoted bacterial clearance after infection with Staphylococcus aureus and, by licensing encephalitogenic Th17 cells, played a key role in the development of autoimmune disease in the central nervous system.

Integrative Analysis of Transcriptomic and Proteomic Profiling in Inflammatory Bowel Disease Colon Biopsies.

BACKGROUND: Crohn's disease (CD) and ulcerative colitis (UC) are intestinal chronic inflammatory conditions characterized by altered epithelial barrier function and tissue damage. Despite significant efforts to understanding the biological mechanisms responsible for gut inflammation, the pathophysiology of CD and UC remains poorly understood. METHODS: To help elucidate the potential mechanisms responsible for gut inflammation in CD and UC, transcriptomic and proteomic profiling of human colon biopsy specimens was performed. Dysregulated genes and proteins in disease tissues compared with normal tissues were characterized from the expression profiles and further subjected to pathway analysis to identify altered biological processes and signaling pathways. RESULTS: Sample analysis showed 4250 genes with matched protein expression and a wide range of correlation of RNA-protein abundance across samples. Pathway analysis of dysregulated genes and proteins in CD and UC showed alterations in immune and inflammatory responses, complement cascade, and the suppression of metabolic processes and PPAR signaling. In CD, increased T-helper cell differentiation and elevated toll-like receptor and JAK/STAT signaling were observed. Interestingly, increased MAPK signaling was only observed in UC. Weighted gene co-expression network analysis suggested a possible role of epigenetic regulation in UC. Of note, a large discrepancy between regulation of RNA and protein levels in inflamed colon samples was detected for previously identified biomarkers including MMP14 and LAMP1. CONCLUSIONS: With the analysis of dysregulated genes and pathways, the present study unravels key mechanisms contributing to CD and UC pathogenesis and emphasizes that integrative analysis of multi-omics data sets can provide more insight into understanding complex disease mechanisms.

Utilization of causal reasoning of hepatic gene expression in rats to identify molecular pathways of idiosyncratic drug-induced liver injury.

Drug-induced liver injury (DILI) represents a leading cause of acute liver failure. Although DILI can be discovered in preclinical animal toxicology studies and/or early clinical trials, some human DILI reactions, termed idiosyncratic DILI (IDILI), are less predictable, occur in a small number of individuals, and do not follow a clear dose-response relationship. The emergence of IDILI poses a critical health challenge for patients and a financial challenge for the pharmaceutical industry. Understanding the cellular and molecular mechanisms underlying IDILI is key to the development of models that can assess potential IDILI risk. This study used Reverse Causal Reasoning (RCR), a method to assess activation of molecular signaling pathways, on gene expression data from rats treated with IDILI or pharmacologic/chemical comparators (NON-DILI) at the maximum tolerated dose to identify mechanistic pathways underlying IDILI. Detailed molecular networks involved in mitochondrial injury, inflammation, and endoplasmic reticulum (ER) stress were found in response to IDILI drugs but not negative controls (NON-DILI). In vitro assays assessing mitochondrial or ER function confirmed the effect of IDILI compounds on these systems. Together our work suggests that using gene expression data can aid in understanding mechanisms underlying IDILI and can guide in vitro screening for IDILI. Specifically, RCR should be considered for compounds that do not show evidence of DILI in preclinical animal studies positive for mitochondrial dysfunction and ER stress assays, especially when the therapeutic index toward projected human maximum drug plasma concentration is low.

Genes associated with intestinal permeability in ulcerative colitis: changes in expression following infliximab therapy.

BACKGROUND: Alterations in intestinal permeability have been implicated in ulcerative colitis (UC). Infliximab, a monoclonal anti-tumor necrosis factor alpha (TNFα) antibody, can induce clinical response in UC. Gene expression in colonic biopsies taken from responders and nonresponders to infliximab can provide insight into the mechanisms of the altered intestinal permeability at a molecular level. METHODS: Colonic biopsies (n = 18 anti-TNFα naïve UC patients; n = 8 normal controls; n = 80 Active Ulcerative Colitis Trial [ACT] 1 patients) were analyzed for mRNA expression using gene expression microarrays. Computational reverse causal reasoning was applied to build causal network models of UC and response and nonresponse of UC to treatment. Quantitative reverse-transcription polymerase chain reaction (qPCR) was used to confirm differentially expressed genes. RESULTS: Reverse causal reasoning on mRNA expression data from anti-TNFα-naïve UC and normal samples provided a mechanistic disease model of the biology of gene expression observed in UC. mRNA expression data from the ACT 1 study enabled construction of a mechanistic model describing the biology of nonresponders to infliximab, including evidence for increased intestinal permeability compared with normal and responder samples. Gene expression changes identified as central to intestinal permeability dysregulation were confirmed in normal, UC, and infliximab-treated patients by qPCR analysis. Gene expression returned toward normal levels in infliximab responders, but not in nonresponders. CONCLUSION: Gene expression analysis and causal network modeling in combination showed that aberrant mRNA expression of genes involved in intestinal epithelial permeability for infliximab responders was restored toward levels observed in normal samples. Infliximab nonresponders showed no equivalent restoration in the expression of these genes.

Interferon-γ plays a role in bone formation in vivo and rescues osteoporosis in ovariectomized mice.

Interferon γ (IFN-γ) is a cytokine produced locally in the bone microenvironment by cells of immune origin as well as mesenchymal stem cells. However, its role in normal bone remodeling is still poorly understood. In this study we first examined the consequences of IFN-γ ablation in vivo in C57BL/6 mice expressing the IFN-γ receptor knockout phenotype (IFNγR1(-/-)). Compared with their wild-type littermates (IFNγR1(+/+)), IFNγR1(-/-) mice exhibit a reduction in bone volume associated with significant changes in cortical and trabecular structural parameters characteristic of an osteoporotic phenotype. Bone histomorphometry of IFNγR1(-/-) mice showed a low-bone-turnover pattern with a decrease in bone formation, a significant reduction in osteoblast and osteoclast numbers, and a reduction in circulating levels of bone-formation and bone-resorption markers. Furthermore, administration of IFN-γ (2000 and 10,000 units) to wild-type C57BL/6 sham-operated (SHAM) and ovariectomized (OVX) female mice significantly improved bone mass and microarchitecture, mechanical properties of bone, and the ratio between bone formation and bone resorption in SHAM mice and rescued osteoporosis in OVX mice. These data therefore support an important physiologic role for IFN-γ signaling as a potential new anabolic therapeutic target for osteoporosis.

Causal reasoning identifies mechanisms of sensitivity for a novel AKT kinase inhibitor, GSK690693.

BACKGROUND: Inappropriate activation of AKT signaling is a relatively common occurrence in human tumors, and can be caused by activation of components of, or by loss or decreased activity of inhibitors of, this signaling pathway. A novel, pan AKT kinase inhibitor, GSK690693, was developed in order to interfere with the inappropriate AKT signaling seen in these human malignancies. Causal network modeling is a systematic computational analysis that identifies upstream changes in gene regulation that can serve as explanations for observed changes in gene expression. In this study, causal network modeling is employed to elucidate mechanisms of action of GSK690693 that contribute to its observed biological effects. The mechanism of action of GSK690693 was evaluated in multiple human tumor cell lines from different tissues in 2-D cultures and xenografts using RNA expression and phosphoproteomics data. Understanding the molecular mechanism of action of novel targeted agents can enhance our understanding of various biological processes regulated by the intended target and facilitate their clinical development. RESULTS: Causal network modeling on transcriptomic and proteomic data identified molecular networks that are comprised of activated or inhibited mechanisms that could explain observed changes in the sensitive cell lines treated with GSK690693. Four networks common to all cell lines and xenografts tested were identified linking GSK690693 inhibition of AKT kinase activity to decreased proliferation. These networks included increased RB1 activity, decreased MYC activity, decreased TFRC activity, and increased FOXO1/FOXO3 activity. CONCLUSION: AKT is involved in regulating both cell proliferation and apoptotic pathways; however, the primary effect with GSK690693 appears to be anti-proliferative in the cell lines and xenografts evaluated. Furthermore, these results indicate that anti-proliferative responses to GSK690693 in either 2-D culture or xenograft models may share common mechanisms within and across sensitive cell lines.

Autocrine regulation of interferon gamma in mesenchymal stem cells plays a role in early osteoblastogenesis.

Interferon (IFN)gamma is a strong inhibitor of osteoclast differentiation and activity. However, its role in osteoblastogenesis has not been carefully examined. Using microarray expression analysis, we found that several IFNgamma-inducible genes were upregulated during early phases of osteoblast differentiation of human mesenchymal stem cells (hMSCs). We therefore hypothesized that IFNgamma may play a role in this process. We first observed a strong and transient increase in IFNgamma production following hMSC induction to differentiate into osteoblasts. We next blocked this endogenous production using a knockdown approach with small interfering RNA and observed a strong inhibition of hMSC differentiation into osteoblasts with a concomitant decrease in Runx2, a factor indispensable for osteoblast development. Additionally, exogenous addition of IFNgamma accelerated hMSC differentiation into osteoblasts in a dose-dependent manner and induced higher levels of Runx2 expression during the early phase of differentiation. We next examined IFNgamma signaling in vivo in IFNgamma receptor 1 knockout (IFNgammaR1(-/-)) mice. Compared with their wild-type littermates, IFNgammaR1(-/-) mice exhibited a reduction in bone mineral density. As in the in vitro experiments, MSCs obtained from IFNgammaR1(-/-) mice showed a lower capacity to differentiate into osteoblasts. In summary, we demonstrate that the presence of IFNgamma plays an important role during the commitment of MSCs into the osteoblastic lineage both in vitro and in vivo, and that this process can be accelerated by exogenous addition of IFNgamma. These data therefore support a new role for IFNgamma as an autocrine regulator of hMSC differentiation and as a potential new target of bone-forming cells in vivo.

Phosphorylation of the human retinoid X receptor alpha at serine 260 impairs coactivator(s) recruitment and induces hormone resistance to multiple ligands.

The retinoid X receptor alpha (RXRalpha) is a member of the nuclear receptor superfamily that regulates transcription of target genes through heterodimerization with several partners, including peroxisome proliferator-activated receptor, retinoic acid receptor, thyroid receptor, and vitamin D receptor (VDR). We have shown previously that signaling through VDR.RXRalpha heterodimers was attenuated in ras-transformed keratinocytes due to phosphorylation of serine 260 of the RXRalpha via the activated Ras-Raf-MAPK cascade in these cells. In this study we demonstrate that phosphorylation at serine 260, a site located in the omega loop-AF-2 interacting domain of RXRalpha, inhibits signaling through several heterodimeric partners of the RXRalpha. The inhibition of signaling results in reduced transactivational response to ligand presentation and the reduced physiological response of growth inhibition not only of 1,25-dihydroxyvitamin D3 but also of retinoic acid receptor alpha ligands and LG1069 (an RXRalpha ligand). This partial resistance to ligands could be reversed by inhibition of MAPK activity or by overexpression of a non-phosphorylable RXRalpha mutant at serine 260 (RXRalpha Ser-260-->Ala). Importantly, phosphorylation of RXRalpha at serine 260 impaired the recruitment of DRIP205 and other coactivators to the VDR.RXRalpha complex. Chromatin immunoprecipitation and pulldown assays further demonstrated that coactivator recruitment to the VDR.RXR complex could be restored by treatment with a MAPK inhibitor. Our data suggest that phosphorylation at serine 260 plays a critical role in inducing hormone resistance of RXRalpha-mediated signaling likely through structural changes in the H1-H3 omega loop-AF2 coactivator(s) interacting domain.

1,25(OH)2D3 acts as a bone-forming agent in the hormone-independent senescence-accelerated mouse (SAM-P/6).

Recent studies suggest that vitamin D signaling regulates bone formation. However, the overall effect of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] on bone turnover in vivo is still unclear. In this study, our aim was to examine the effect of 1,25(OH)2D3 on bone turnover in SAM-P/6, a hormone-independent mouse model of senile osteoporosis characterized by a decrease in bone formation. Male and female 4-mo-old SAM-P/6 mice were treated with 1,25(OH)2D3 (18 pmol/24 h) or vehicle for a period of 6 wk, and a group of age- and sex-matched nonosteoporotic animals was used as control. Bone mineral density (BMD) at the lumbar spine increased rapidly by >30 +/- 5% (P < 0.001) in 1,25(OH)2D3-treated SAM-P/6 animals, whereas BMD decreased significantly by 18 +/- 2% (P < 0.01) in vehicle-treated SAM-P/6 animals and remained stable in control animals during the same period. Static and dynamic bone histomorphometry indicated that 1,25(OH)2D3 significantly increased bone volume and other parameters of bone quality as well as subperiosteal bone formation rate compared with vehicle-treated SAM-P/6 mice. However, no effect on trabecular bone formation was observed. This was accompanied by a marked decrease in the number of osteoclasts and eroded surfaces. A significant increase in circulating bone formation markers and a decrease in bone resorption markers was also observed. Finally, bone marrow cells, obtained from 1,25(OH)2D3-treated animals and cultured in the absence of 1,25(OH)2D3, differentiated more intensely into osteoblasts compared with those derived from vehicle-treated mice cultured in the same conditions. Taken together, these findings demonstrate that 1,25(OH)2D3 acts simultaneously on bone formation and resorption to prevent the development of senile osteoporosis.

Vitamin D treatment of senescence accelerated mice (SAM-P/6) induces several regulators of stromal cell plasticity.

In an attempt to understand the regulation of bone marrow multipotential cells plasticity in vivo, we treated 4-month-old SAM-P/6 mice with a constant infusion of either 18 pmol/24 h of 1,25(OH)2D3 or vehicle alone for 6 weeks. In vehicle treated animals 78% +/- 4 adipose volume vs. total volume was stained positive with oil red O as compared to only 32 +/- 3% in 1,25(OH)2D3 treated animals (P < 0.001). Furthermore, we aimed to identify the changes in gene expression induced by 1,25(OH)2D3 in bone marrow cells by analyzing a set of 5440 genes in the NIA 15K Mouse cDNA microarray. Overall, a coordinated regulation of genes which both stimulate osteoblastogenesis and inhibit adipogenesis was observed in 1,25(OH)2D3-treated mice when compared to vehicle treated mice. In summary, this study illustrates the anti-adipogenic effect of 1,25(OH)2D3 in bone cells and identifies some of the possible key signals involved in bone cell plasticity.

1,25(OH)2D3 inhibits bone marrow adipogenesis in senescence accelerated mice (SAM-P/6) by decreasing the expression of peroxisome proliferator-activated receptor gamma 2 (PPARgamma2).

Senile osteoporosis is the endpoint of a continuum that starts after the third decade of life when peak bone mass is attained and then is followed by a progressive and irreversible decline in bone mass. One of the mechanisms that could explain this is the increasing levels of adipogenesis in bone marrow seen with increasing age, probably due to alterations in the differentiation of mesenchymal stem cells (MSC). Senescence accelerated mice (SAM-P/6) constitute an accepted model for senile osteoporosis since their loss of bone mineral density is clearly due to high levels of adipogenesis and a deficit in osteoblastogenesis. It is known that MSC expressing a ligand-activated transcription factor known as peroxisome proliferators-activated receptor gamma 2 (PPARgamma2) are committed to differentiate into adipocytes. The regulation of PPARgamma2 activation may play a role in the control of adipogenic differentiation of MSC and thus contribute to their differentiation into osteoblasts in order to form new bone. Our previous studies have shown that the active form of vitamin D (1,25(OH)(2)D(3)) plays a role as a bone forming agent because it induces osteoblastogenesis and inhibits adipogenesis in bone marrow of SAM-P/6 mice. To elucidate the role of 1,25(OH)(2)D(3) on the expression of PPARgamma2 we treated 4-month old SAM-P/6 mice with 1,25(OH)(2)D(3) (18pmol/24 h) or vehicle during 6 weeks. Initially we found that with aging the levels of PPARgamma2 expression increase in bone marrow of SAM-P/6 (P<0.001) We then measured the changes in the expression of PPARgamma2 by semi-quantitative reverse transcription-polymerase chain reaction and immunofluorescence. We found a significant reduction of PPARgamma2-expressing cells in 1,25(OH)(2)D(3)-treated (32% +/-6) as compared to vehicle (76% +/-5) treated mice (p<0.01) In summary, this study shows that the administration of 1,25(OH)(2)D(3) in an in vivo model of senile osteoporosis is associated with reduction in PPARgamma2 a key transcription factor for the adipose differentiation of MSC.

Estrogens (E2) regulate expression and response of 1,25-dihydroxyvitamin D3 receptors in bone cells: changes with aging and hormone deprivation.

Studies on the effect of estrogens (E(2)) on the expression of vitamin D receptor (VDR) and its bioresponse in bone have demonstrated that E(2) modulate activity and increase the number of VDRs in vitro; however, no in vivo studies have been pursued to assess this interaction. Our study identifies the changes in the number of VDR-expressing cells in bone of C57BL/6J young and old oophorectomized mice (4 and 24 months) with and without 17beta estradiol (E(2)) replacement. A total of 36 mice were sacrificed; both tibiae and femora were isolated and VDR expression was quantified by Northern blot, immunohistochemistry, immunofluorescence, and flow cytometry. Among the intact mice there was a significant difference in the number of VDR-expressing osteoblasts between young (68%) and old (56%) (p<0.04). In young oophorectomized mice the number of VDR-expressing osteoblasts decreased from 68% to 46% after oophorectomy and recovered to 72% after E(2) administration (p<0.02), while in the group of old mice, the number of VDR-expressing osteoblasts decreased from 56% to 48% after oophorectomy (p<0.01) and recovered to 85% after E(2) administration (p<0.001). Our results show that VDR expression in bone decreases with aging and estrogen deprivation but recovers after E(2) supplementation in both young and old mice with a more significant level of response in older bone. To evaluate the level of VDR bioresponse to E(2) we assessed the effect of E(2) supplementation to human osteoblasts (N-976) in vitro. Northern blot showed a significant up-regulation of VDR expression in E(2) treated cells as compared to non-treated cells (p<0.05). We also assessed the previously known anti-apoptotic effect of vitamin D in osteoblasts in vitro after serum deprivation by using either E(2), E(2)+1,25(OH)(2)D(3), or 1,25(OH)(2)D(3) alone. We found a lower number of apoptotic cells and longer cell survival after 48 h of treatment with 1,25(OH)(2)D(3)+E(2) as compared to 1,25(OH)(2)D(3) or E(2) alone (p<0.002). In summary, our results demonstrate that E(2) increases VDR expression in bone in vivo and potentiate the bioresponse of VDR in osteoblasts in vitro.