CXCL10 deficiency limits macrophage infiltration, preserves lung matrix, and enables lung growth in bronchopulmonary dysplasia.

Preterm infants with oxygen supplementation are at high risk for bronchopulmonary dysplasia (BPD), a neonatal chronic lung disease. Inflammation with macrophage activation is central to the pathogenesis of BPD. CXCL10, a chemotactic and pro-inflammatory chemokine, is elevated in the lungs of infants evolving BPD and in hyperoxia-based BPD in mice. Here, we tested if CXCL10 deficiency preserves lung growth after neonatal hyperoxia by preventing macrophage activation. To this end, we exposed Cxcl10 knockout (Cxcl10-/-) and wild-type mice to an experimental model of hyperoxia (85% O2)-induced neonatal lung injury and subsequent regeneration. In addition, cultured primary human macrophages and murine macrophages (J744A.1) were treated with CXCL10 and/or CXCR3 antagonist. Our transcriptomic analysis identified CXCL10 as a central hub in the inflammatory network of neonatal mouse lungs after hyperoxia. Quantitative histomorphometric analysis revealed that Cxcl10-/- mice are in part protected from reduced alveolar. These findings were related to the preserved spatial distribution of elastic fibers, reduced collagen deposition, and protection from macrophage recruitment/infiltration to the lungs in Cxcl10-/- mice during acute injury and regeneration. Complimentary, studies with cultured human and murine macrophages showed that hyperoxia induces Cxcl10 expression that in turn triggers M1-like activation and migration of macrophages through CXCR3. Finally, we demonstrated a temporal increase of macrophage-related CXCL10 in the lungs of infants with BPD. In conclusion, our data demonstrate macrophage-derived CXCL10 in experimental and clinical BPD that drives macrophage chemotaxis through CXCR3, causing pro-fibrotic lung remodeling and arrest of alveolarization. Thus, targeting the CXCL10-CXCR3 axis could offer a new therapeutic avenue for BPD.

Myeloperoxidase is a critical mediator of anthracycline-induced cardiomyopathy.

Cardiotoxicity is a major complication of anthracycline therapy that negatively impacts prognosis. Effective pharmacotherapies for prevention of anthracycline-induced cardiomyopathy (AICM) are currently lacking. Increased plasma levels of the neutrophil-derived enzyme myeloperoxidase (MPO) predict occurrence of AICM in humans. We hypothesized that MPO release causally contributes to AICM. Mice intravenously injected with the anthracycline doxorubicin (DOX) exhibited higher neutrophil counts and MPO levels in the circulation and cardiac tissue compared to saline (NaCl)-treated controls. Neutrophil-like HL-60 cells exhibited increased MPO release upon exposition to DOX. DOX induced extensive nitrosative stress in cardiac tissue alongside with increased carbonylation of sarcomeric proteins in wildtype but not in Mpo-/- mice. Accordingly, co-treatment of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) with DOX and MPO aggravated loss of hiPSC-CM-contractility compared to DOX treatment alone. DOX-treated animals exhibited pronounced cardiac apoptosis and inflammation, which was attenuated in MPO-deficient animals. Finally, genetic MPO deficiency and pharmacological MPO inhibition protected mice from the development of AICM. The anticancer efficacy of DOX was unaffected by MPO deficiency. Herein we identify MPO as a critical mediator of AICM. We demonstrate that DOX induces cardiac neutrophil infiltration and release of MPO, which directly impairs cardiac contractility through promoting oxidation of sarcomeric proteins, cardiac inflammation and cardiomyocyte apoptosis. MPO thus emerges as a promising pharmacological target for prevention of AICM.

Functional impact and molecular binding modes of drugs that target the PI3K isoform p110δ.

Targeting the PI3K isoform p110δ against B cell malignancies is at the mainstay of PI3K inhibitor (PI3Ki) development. Therefore, we generated isogenic cell lines, which express wild type or mutant p110δ, for assessing the potency, isoform-selectivity and molecular interactions of various PI3Ki chemotypes. The affinity pocket mutation I777M maintains p110δ activity in the presence of idelalisib, as indicated by intracellular AKT phosphorylation, and rescues cell functions such as p110δ-dependent cell viability. Resistance owing to this substitution consistently affects the potency of p110δ-selective in contrast to most multi-targeted PI3Ki, thus distinguishing usually propeller-shaped and typically flat molecules. Accordingly, molecular dynamics simulations indicate that the I777M substitution disturbs conformational flexibility in the specificity or affinity pockets of p110δ that is necessary for binding idelalisib or ZSTK474, but not copanlisib. In summary, cell-based and molecular exploration provide comparative characterization of currently developed PI3Ki and structural insights for future PI3Ki design.

Transcriptomic profiles and 5-year results from the randomized CLL14 study of venetoclax plus obinutuzumab versus chlorambucil plus obinutuzumab in chronic lymphocytic leukemia.

Data on long-term outcomes and biological drivers associated with depth of remission after BCL2 inhibition by venetoclax in the treatment of chronic lymphocytic leukemia (CLL) are limited. In this open-label parallel-group phase-3 study, 432 patients with previously untreated CLL were randomized (1:1) to receive either 1-year venetoclax-obinutuzumab (Ven-Obi, 216 patients) or chlorambucil-Obi (Clb-Obi, 216 patients) therapy (NCT02242942). The primary endpoint was investigator-assessed progression-free survival (PFS); secondary endpoints included minimal residual disease (MRD) and overall survival. RNA sequencing of CD19-enriched blood was conducted for exploratory post-hoc analyses. After a median follow-up of 65.4 months, PFS is significantly superior for Ven-Obi compared to Clb-Obi (Hazard ratio [HR] 0.35 [95% CI 0.26-0.46], p < 0.0001). At 5 years after randomization, the estimated PFS rate is 62.6% after Ven-Obi and 27.0% after Clb-Obi. In both arms, MRD status at the end of therapy is associated with longer PFS. MRD + ( ≥ 10-4) status is associated with increased expression of multi-drug resistance gene ABCB1 (MDR1), whereas MRD6 (< 10-6) is associated with BCL2L11 (BIM) expression. Inflammatory response pathways are enriched in MRD+ patient solely in the Ven-Obi arm. These data indicate sustained long-term efficacy of fixed-duration Ven-Obi in patients with previously untreated CLL. The distinct transcriptomic profile of MRD+ status suggests possible biological vulnerabilities.

The proto-oncogene TCL1A deregulates cell cycle and genomic stability in CLL.

Upregulation of the proto-oncogene T-cell leukemia/lymphoma 1A (TCL1A) is causally implicated in various B-cell and T-cell malignancies. High-level TCL1A correlates with aggressive disease features and inferior clinical outcomes. However, the molecular and cell biological consequences of, particularly nuclear, TCL1A are not fully elucidated. We observed here in mouse models of subcellular site-specific TCL1A-induced lymphomagenesis that TCL1A exerts a strong transforming impact via nuclear topography. In proteomic screens of TCL1A-bound molecules in chronic lymphocytic leukemia (CLL) cells and B-cell lymphoma lines, we identified regulators of cell cycle and DNA repair pathways as novel TCL1A interactors, particularly enriched under induced DNA damage and mitosis. By functional mapping and in silico modeling, we specifically identified the mitotic checkpoint protein, cell division cycle 20 (CDC20), as a direct TCL1A interactor. According to the regulatory impact of TCL1A on the activity of the CDC20-containing mitotic checkpoint and anaphase-promoting complexes during mitotic progression, TCL1A overexpression accelerated cell cycle transition in B-cell lymphoma lines, impaired apoptotic damage responses in association with pronounced chromosome missegregation, and caused cellular aneuploidy in Eµ-TCL1A mice. Among hematopoietic cancers, CDC20 levels seem particularly low in CLL. CDC20 expression negatively correlated with TCL1A and lower expression marked more aggressive and genomically instable disease and cellular phenotypes. Knockdown of Cdc20 in TCL1A-initiated murine CLL promoted aneuploidy and leukemic acceleration. Taken together, we discovered a novel cell cycle-associated effect of TCL1A abrogating controlled cell cycle transition. This adds to our concept of oncogenic TCL1A by targeting genome stability. Overall, we propose that TCL1A acts as a pleiotropic adapter molecule with a synergistic net effect of multiple hijacked pathways.

The scaffold protein NEDD9 is necessary for leukemia-cell migration and disease progression in a mouse model of chronic lymphocytic leukemia.

The scaffold protein NEDD9 is frequently upregulated and hyperphosphorylated in cancers, and is associated with poor clinical outcome. NEDD9 promotes B-cell adhesion, migration and chemotaxis, pivotal processes for malignant development. We show that global or B-cell-specific deletion of Nedd9 in chronic lymphocytic leukemia (CLL) mouse models delayed CLL development, markedly reduced disease burden and resulted in significant survival benefit. NEDD9 was required for efficient CLL cell homing, chemotaxis, migration and adhesion. In CLL patients, peripheral NEDD9 expression was associated with adhesion and migration signatures as well as leukocyte count. Additionally, CLL lymph nodes frequently expressed high NEDD9 levels, with a subset of patients showing NEDD9 expression enriched in the CLL proliferation centers. Blocking activity of prominent NEDD9 effectors, including AURKA and HDAC6, effectively reduced CLL cell migration and chemotaxis. Collectively, our study provides evidence for a functional role of NEDD9 in CLL pathogenesis that involves intrinsic defects in adhesion, migration and homing.

COVID-19 Infection Induce miR-371a-3p Upregulation Resulting in Influence on Male Fertility.

In December 2019, the first case of COVID-19 was reported and since then several groups have already published that the virus can be present in the testis. To study the influence of SARS-CoV-2 which cause a dysregulation of the androgen receptor (AR) level, thereby leading to fertility problems and inducing germ cell testicular changes in patients after the infection. Formalin-Fixed-Paraffin-Embedded (FFPE) testicular samples from patients who died with or as a result of COVID-19 (n = 32) with controls (n = 6), inflammatory changes (n = 9), seminoma with/without metastasis (n = 11) compared with healthy biopsy samples (n = 3) were analyzed and compared via qRT-PCR for the expression of miR-371a-3p. An immunohistochemical analysis (IHC) and ELISA were performed in order to highlight the miR-371a-3p targeting the AR. Serum samples of patients with mild or severe COVID-19 symptoms (n = 34) were analyzed for miR-371a-3p expression. In 70% of the analyzed postmortem testicular tissue samples, a significant upregulation of the miR-371a-3p was detected, and 75% of the samples showed a reduced spermatogenesis. In serum samples, the upregulation of the miR-371a-3p was also detectable. The upregulation of the miR-371a-3p is responsible for the downregulation of the AR in SARS-CoV-2-positive patients, resulting in decreased spermatogenesis. Since the dysregulation of the AR is associated with infertility, further studies have to confirm if the identified dysregulation is regressive after a declining infection.

Extracellular vesicles and PD-L1 suppress macrophages, inducing therapy resistance in TP53-deficient B-cell malignancies.

Genetic alterations in the DNA damage response (DDR) pathway are a frequent mechanism of resistance to chemoimmunotherapy (CIT) in B-cell malignancies. We have previously shown that the synergy of CIT relies on secretory crosstalk elicited by chemotherapy between the tumor cells and macrophages. Here, we show that loss of multiple different members of the DDR pathway inhibits macrophage phagocytic capacity in vitro and in vivo. Particularly, loss of TP53 led to decreased phagocytic capacity ex vivo across multiple B-cell malignancies. We demonstrate via in vivo cyclophosphamide treatment using the Eµ-TCL1 mouse model that loss of macrophage phagocytic capacity in Tp53-deleted leukemia is driven by a significant downregulation of a phagocytic transcriptomic signature using small conditional RNA sequencing. By analyzing the tumor B-cell proteome, we identified a TP53-specific upregulation of proteins associated with extracellular vesicles (EVs). We abrogated EV biogenesis in tumor B-cells via clustered regularly interspaced short palindromic repeats (CRISPR)-knockout (KO) of RAB27A and confirmed that the EVs from TP53-deleted lymphoma cells were responsible for the reduced phagocytic capacity and the in vivo CIT resistance. Furthermore, we observed that TP53 loss led to an upregulation of both PD-L1 cell surface expression and secretion of EVs by lymphoma cells. Disruption of EV bound PD-L1 by anti-PD-L1 antibodies or PD-L1 CRISPR-KO improved macrophage phagocytic capacity and in vivo therapy response. Thus, we demonstrate enhanced EV release and increased PD-L1 expression in TP53-deficient B-cell lymphomas as novel mechanisms of macrophage function alteration in CIT resistance. This study indicates the use of checkpoint inhibition in the combination treatment of B-cell malignancies with TP53 loss.

Macrophage-derived IL-6 trans-signalling as a novel target in the pathogenesis of bronchopulmonary dysplasia.

RATIONALE: Premature infants exposed to oxygen are at risk for bronchopulmonary dysplasia (BPD), which is characterised by lung growth arrest. Inflammation is important, but the mechanisms remain elusive. Here, we investigated inflammatory pathways and therapeutic targets in severe clinical and experimental BPD. METHODS AND RESULTS: First, transcriptomic analysis with in silico cellular deconvolution identified a lung-intrinsic M1-like-driven cytokine pattern in newborn mice after hyperoxia. These findings were confirmed by gene expression of macrophage-regulating chemokines (Ccl2, Ccl7, Cxcl5) and markers (Il6, Il17A, Mmp12). Secondly, hyperoxia-activated interleukin 6 (IL-6)/signal transducer and activator of transcription 3 (STAT3) signalling was measured in vivo and related to loss of alveolar epithelial type II cells (ATII) as well as increased mesenchymal marker. Il6 null mice exhibited preserved ATII survival, reduced myofibroblasts and improved elastic fibre assembly, thus enabling lung growth and protecting lung function. Pharmacological inhibition of global IL-6 signalling and IL-6 trans-signalling promoted alveolarisation and ATII survival after hyperoxia. Third, hyperoxia triggered M1-like polarisation, possibly via Krüppel-like factor 4; hyperoxia-conditioned medium of macrophages and IL-6-impaired ATII proliferation. Finally, clinical data demonstrated elevated macrophage-related plasma cytokines as potential biomarkers that identify infants receiving oxygen at increased risk of developing BPD. Moreover, macrophage-derived IL6 and active STAT3 were related to loss of epithelial cells in BPD lungs. CONCLUSION: We present a novel IL-6-mediated mechanism by which hyperoxia activates macrophages in immature lungs, impairs ATII homeostasis and disrupts elastic fibre formation, thereby inhibiting lung growth. The data provide evidence that IL-6 trans-signalling could offer an innovative pharmacological target to enable lung growth in severe neonatal chronic lung disease.

Kinase activity profiling reveals contribution of G-protein signaling modulator 2 deficiency to impaired regulatory T cell migration in rheumatoid arthritis.

The ability of regulatory T (Treg) cells to migrate into inflammatory sites is reduced in autoimmune diseases, including rheumatoid arthritis (RA). The reasons for impaired Treg cell migration remain largely unknown. We performed multiplex human kinase activity arrays to explore possible differences in the post-translational phosphorylation status of kinase related proteins that could account for altered Treg cell migration in RA. Results were verified by migration assays and Western blot analysis of CD4+ T cells from RA patients and from mice with collagen type II induced arthritis. Kinome profiling of CD4+ T cells from RA patients revealed significantly altered post-translational phosphorylation of kinase related proteins, including G-protein-signaling modulator 2 (GPSM2), protein tyrosine kinase 6 (PTK6) and vitronectin precursor (VTNC). These proteins have not been associated with RA until now. We found that GPSM2 expression is reduced in CD4+ T cells from RA patients and is significantly downregulated in experimental autoimmune arthritis following immunization of mice with collagen type II. Interestingly, GPSM2 acts as a promoter of Treg cell migration in healthy individuals. Treatment of RA patients with interleukin-6 receptor (IL-6R) blocking antibodies restores GPSM2 expression, thereby improving Treg cell migration. Our study highlights the potential of multiplex kinase activity arrays as a tool for the identification of RA-related proteins which could serve as targets for novel treatments.

Active Akt signaling triggers CLL toward Richter transformation via overactivation of Notch1.

Richter's transformation (RT) is an aggressive lymphoma that occurs upon progression from chronic lymphocytic leukemia (CLL). Transformation has been associated with genetic aberrations in the CLL phase involving TP53, CDKN2A, MYC, and NOTCH1; however, a significant proportion of RT cases lack CLL phase-associated events. Here, we report that high levels of AKT phosphorylation occur both in high-risk CLL patients harboring TP53 and NOTCH1 mutations as well as in patients with RT. Genetic overactivation of Akt in the murine Eµ-TCL1 CLL mouse model resulted in CLL transformation to RT with significantly reduced survival and an aggressive lymphoma phenotype. In the absence of recurrent mutations, we identified a profile of genomic aberrations intermediate between CLL and diffuse large B-cell lymphoma. Multiomics assessment by phosphoproteomic/proteomic and single-cell transcriptomic profiles of this Akt-induced murine RT revealed an S100 protein-defined subcluster of highly aggressive lymphoma cells that developed from CLL cells, through activation of Notch via Notch ligand expressed by T cells. Constitutively active Notch1 similarly induced RT of murine CLL. We identify Akt activation as an initiator of CLL transformation toward aggressive lymphoma by inducing Notch signaling between RT cells and microenvironmental T cells.

Inflammatory Pre-Conditioning of Adipose-Derived Stem Cells with Cerebrospinal Fluid from Traumatic Brain Injury Patients Alters the Immunomodulatory Potential of ADSC Secretomes.

Immunomodulation by adipose-tissue-derived stem cells (ADSCs) is of special interest for the alleviation of damaging inflammatory responses in central nervous system injuries. The present study explored the effects of cerebrospinal fluid (CSF) from traumatic brain injury (TBI) patients on this immunomodulatory potential of ADSCs. CSF conditioning of ADSCs increased messenger RNA levels of both pro- and anti-inflammatory genes compared to controls. Exposure of phorbol-12-myristate-13-acetate-differentiated THP1 macrophages to the secretome of CSF-conditioned ADSCs downregulated both proinflammatory (cyclooxygenase-2, tumor necrosis factor alpha) and anti-inflammatory (suppressor of cytokine signaling 3, interleukin-1 receptor antagonist, and transforming growth factor beta) genes in these cells. Interleukin-10 expression was elevated in both naïve and conditioned secretomes. ADSC secretome treatment, further, induced macrophage maturation of THP1 cells and increased the percentage of CD11b+, CD14+, CD86+, and, to a lesser extent, CD206+ cells. This, moreover, enhanced the phagocytic activity of CD14+ and CD86+ cells, though independently of pre-conditioning. Secretome exposure, finally, also induced a reduction in the percentage of CD192+ adherent cells in cultures of peripheral blood mononuclear cells (PBMCs) from both healthy subjects and TBI patients. This limited efficacy (of both naïve and pre-conditioned secretomes) suggests that the effects of lymphocyte-monocyte paracrine signaling on the fate of cultured PBMCs are strongest upon adherent cell populations.

Macrophage-Mediated Antibody Dependent Effector Function in Aggressive B-Cell Lymphoma Treatment is Enhanced by Ibrutinib via Inhibition of JAK2.

Targeted inhibition of Bruton's Tyrosine Kinase (BTK) with ibrutinib and other agents has become important treatment options in chronic lymphocytic leukemia, Waldenström's Macroglobulinemia, Mantle cell lymphoma, and non-GCB DLBCL. Clinical trials combining small molecule inhibitors with monoclonal antibodies have been initiated at rapid pace, with the biological understanding between their synergistic interactions lagging behind. Here, we have evaluated the synergy between BTK inhibitors and monoclonal antibody therapy via macrophage mediated antibody dependent cellular phagocytosis (ADCP). Initially, we observed increased ADCP with ibrutinib, whilst second generation BTK inhibitors failed to synergistically interact with monoclonal antibody treatment. Kinase activity profiling under BTK inhibition identified significant loss of Janus Kinase 2 (JAK2) only under ibrutinib treatment. We validated this potential off-target effect via JAK inhibition in vitro as well as with CRISPR/Cas9 JAK2-/- experiments in vivo, showing increased ADCP and prolonged survival, respectively. This data supports inhibition of the JAK-STAT (Signal Transducers and Activators of Transcription) signaling pathway in B-cell malignancies in combination with monoclonal antibody therapy to increase macrophage-mediated immune responses.

Tumor Metabolism as a Regulator of Tumor-Host Interactions in the B-Cell Lymphoma Microenvironment-Fueling Progression and Novel Brakes for Therapy.

Tumor metabolism and its specific alterations have become an integral part of understanding functional alterations leading to malignant transformation and maintaining cancer progression. Here, we review the metabolic changes in B-cell neoplasia, focusing on the effects of tumor metabolism on the tumor microenvironment (TME). Particularly, innate and adaptive immune responses are regulated by metabolites in the TME such as lactate. With steadily increasing therapeutic options implicating or utilizing the TME, it has become essential to address the metabolic alterations in B-cell malignancy for therapeutic approaches. In this review, we discuss metabolic alterations of B-cell lymphoma, consequences for currently used therapy regimens, and novel approaches specifically targeting metabolism in the TME.

Mechanisms of Lymphoma Clearance Induced by High-Dose Alkylating Agents.

The extraordinary activity of high-dose cyclophosphamide against some high-grade lymphomas was described nearly 60 years ago. Here we address mechanisms that mediate cyclophosphamide activity in bona fide human double-hit lymphoma. We show that antibody resistance within the bone marrow (BM) is not present upon early engraftment but develops during lymphoma progression. This resistance required a high tumor:macrophage ratio, was recapitulated in spleen by partial macrophage depletion, and was overcome by multiple, high-dose alkylating agents. Cyclophosphamide induced endoplasmic reticulum (ER) stress in BM-resident lymphoma cells in vivo that resulted in ATF4-mediated paracrine secretion of VEGFA, massive macrophage infiltration, and clearance of alemtuzumab-opsonized cells. BM macrophages isolated after cyclophosphamide treatment had increased phagocytic capacity that was reversed by VEGFA blockade or SYK inhibition. Single-cell RNA sequencing of these macrophages identified a "super-phagocytic" subset that expressed CD36/FCGR4. Together, these findings define a novel mechanism through which high-dose alkylating agents promote macrophage-dependent lymphoma clearance. SIGNIFICANCE: mAbs are effective against only a small subset of cancers. Herein, we recapitulate compartment-specific antibody resistance and define an ER stress-dependent mechanism induced by high-dose alkylating agents that promotes phagocytosis of opsonized tumor cells. This approach induces synergistic effects with mAbs and merits testing across additional tumor types.See related commentary by Duval and De Palma, p. 834.This article is highlighted in the In This Issue feature, p. 813.

Corticosteroids inhibit Mycobacterium tuberculosis-induced necrotic host cell death by abrogating mitochondrial membrane permeability transition.

Corticosteroids are host-directed drugs with proven beneficial effect on survival of tuberculosis (TB) patients, but their precise mechanisms of action in this disease remain largely unknown. Here we show that corticosteroids such as dexamethasone inhibit necrotic cell death of cells infected with Mycobacterium tuberculosis (Mtb) by facilitating mitogen-activated protein kinase phosphatase 1 (MKP-1)-dependent dephosphorylation of p38 MAPK. Characterization of infected mixed lineage kinase domain-like (MLKL) and tumor necrosis factor receptor 1 (TNFR1) knockout cells show that the underlying mechanism is independent from TNFα-signaling and necroptosis. Our results link corticosteroid function and p38 MAPK inhibition to abrogation of necrotic cell death mediated by mitochondrial membrane permeability transition, and open new avenues for research on novel host-directed therapies (HDT).