Immunomodulatory and clinical effects of receptor-interacting protein kinase 1 (RIPK1) inhibitor eclitasertib (SAR443122) in patients with severe COVID-19: a phase 1b, randomized, double-blinded, placebo-controlled study.

BACKGROUND: Targeting receptor-interacting serine/threonine protein kinase 1 could mitigate the devastating sequelae of the hyperinflammatory state observed in severe cases of COVID-19. This study explored the immunomodulatory and clinical effects of the receptor-interacting serine/threonine protein kinase 1 inhibitor SAR443122 (eclitasertib) in patients with severe COVID-19. METHODS: In this Phase 1b, double-blinded, placebo-controlled study (NCT04469621) a total of 82 patients were screened, of whom 68 patients were eligible and randomized (2:1) to receive eclitasertib 600 mg (300 mg twice daily) or placebo up to 14 days. Primary outcome was relative change in C-reactive protein from baseline to Day 7. Time to clinical improvement using 7-point ordinal scale, ventilator/respiratory failure-free days, change in SpO2/FiO2 ratio, and biomarkers of severe COVID-19 were explored. RESULTS: Geometric mean ratio (point estimate [90% confidence interval]) of the relative change from baseline in C-reactive protein with eclitasertib vs. placebo on Day 7 was 0.85 (0.49-1.45; p = 0.30). Median time to 50% decrease in C-reactive protein from baseline was 3 days vs. 5 days (p = 0.056) with eclitasertib vs. placebo. Median time to ≥ 2-point improvement on 7-point clinical symptoms scale was 8 days vs. 10 days with eclitasertib vs. placebo (p = 0.38). Mean ventilator/respiratory failure-free days, change in baseline-adjusted SpO2/FiO2 ratio, and clinical biomarkers showed consistent numerical improvements with eclitasertib vs. placebo. The most frequently reported treatment-emergent adverse events were gastrointestinal disorders and condition aggravated/worsened COVID-19 pneumonia. CONCLUSIONS: Eclitasertib was well tolerated with consistent trends toward more rapid resolution of inflammatory biomarkers and clinical improvement in severe COVID-19 patients than placebo. GOV IDENTIFIER: NCT04469621, first posted on clinicaltrials.gov on July 14, 2020.

Protein kinase D controls voluntary-running-induced skeletal muscle remodelling.

Skeletal muscle responds to exercise by activation of signalling pathways that co-ordinate gene expression to sustain muscle performance. MEF2 (myocyte enhancer factor 2)-dependent transcriptional activation of MHC (myosin heavy chain) genes promotes the transformation from fast-twitch into slow-twitch fibres, with MEF2 activity being tightly regulated by interaction with class IIa HDACs (histone deacetylases). PKD (protein kinase D) is known to directly phosphorylate skeletal muscle class IIa HDACs, mediating their nuclear export and thus derepression of MEF2. In the present study, we report the generation of transgenic mice with inducible conditional expression of a dominant-negative PKD1kd (kinase-dead PKD1) protein in skeletal muscle to assess the role of PKD in muscle function. In control mice, long-term voluntary running experiments resulted in a switch from type IIb+IId/x to type IIa plantaris muscle fibres as measured by indirect immunofluorescence of MHCs isoforms. In mice expressing PKD1kd, this fibre type switch was significantly impaired. These mice exhibited altered muscle fibre composition and decreased running performance compared with control mice. Our findings thus indicate that PKD activity is essential for exercise-induced MEF2-dependent skeletal muscle remodelling in vivo.

Shaping of terminal megakaryocyte differentiation and proplatelet development by sphingosine-1-phosphate receptor S1P4.

Megakaryocytes, which mature from hematopoietic progenitors in the bone marrow, further differentiate by reorganizing their cytoplasm into long proplatelet extensions that release platelets into the circulation. The molecular mechanisms underlying this highly dynamic cytoplasmic and cytoskeletal remodeling process are only poorly understood. Here we report that sphingosine 1-phosphate receptor 4 (S1P(4)) is specifically up-regulated during the development of human megakaryocytes from progenitor cells and is expressed in mature murine megakaryocytes. Megakaryocytes generated from S1P(4)-deficient murine bone marrow showed atypical and reduced formation of proplatelets in vitro. The recovery of platelet numbers after experimental thrombocytopenia was significantly delayed in S1p4(-/-) mice. Remarkably, overexpression and stimulation of S1P(4) in human erythroleukemia HEL cells promoted endomitosis, formation of cytoplasmic extensions, and subsequent release of platelet-like particles. These observations indicate that S1P(4) is involved in shaping the terminal differentiation of megakaryocytes.

IL-23 stimulates epidermal hyperplasia via TNF and IL-20R2-dependent mechanisms with implications for psoriasis pathogenesis.

Aberrant cytokine expression has been proposed as an underlying cause of psoriasis, although it is unclear which cytokines play critical roles. Interleukin (IL)-23 is expressed in human psoriasis and may be a master regulator cytokine. Direct intradermal administration of IL-23 in mouse skin, but not IL-12, initiates a tumor necrosis factor-dependent, but IL-17A-independent, cascade of events resulting in erythema, mixed dermal infiltrate, and epidermal hyperplasia associated with parakeratosis. IL-23 induced IL-19 and IL-24 expression in mouse skin, and both genes were also elevated in human psoriasis. IL-23-dependent epidermal hyperplasia was observed in IL-19-/- and IL-24-/- mice, but was inhibited in IL-20R2-/- mice. These data implicate IL-23 in the pathogenesis of psoriasis and support IL-20R2 as a novel therapeutic target.

Neutrophils regulate airway responses in a model of fungal allergic airways disease.

Neutrophils infiltrate airway walls in patients with allergic airway diseases and in animal models of these illnesses, but their contribution to the pathogenesis of airway allergy is not established. We hypothesized that, in a mouse model of airway allergy to the ubiquitous environmental mold, Aspergillus fumigatus, airway neutrophils contribute to disease severity. Ab-mediated neutrophil depletion resulted in reduced airway hyperresponsiveness and remodeling, whereas conditional transgenic overexpression of the neutrophil chemotactic molecule, CXCL1, in airway walls resulted in worsened allergic responses. This worsened phenotype was associated with a marked increase in the number of airway neutrophils but not other lung leukocytes, including eosinophils and lymphocyte subsets, and depletion of neutrophils in sensitized mice with transgenic overexpression of CXCL1 resulted in attenuated airway responses. The number of lung neutrophils correlated with lung matrix metalloproteinase 9 (MMP-9) activity both in the context of neutrophil depletion and with augmented neutrophil recruitment to the airways. Although wild-type and MMP-9-deficient neutrophils homed to the inflamed airways to a similar extent, transfer of wild-type, but not MMP-9-deficient, neutrophils to MMP-9-deficient animals resulted in augmented allergic airway responses. Taken together, these data implicate neutrophils in the pathogenesis of fungal allergic airway disease.

The chemokine receptor D6 limits the inflammatory response in vivo.

How the inflammatory response is initiated has been well defined but relatively little is known about how such responses are resolved. Here we show that the D6 chemokine receptor is involved in the post-inflammatory clearance of beta-chemokines from cutaneous sites. After induction of inflammation by phorbol esters, wild-type mice showed a transient inflammatory response. However, in D6-deficient mice, an excess concentration of residual chemokines caused a notable inflammatory pathology with similarities to human psoriasis. These results suggest that D6 is involved in the resolution of the cutaneous inflammatory response.

The pyrimidinergic P2Y6 receptor mediates a novel release of proinflammatory cytokines and chemokines in monocytic cells stimulated with UDP.

The human P2Y6 receptor (hP2Y6) is a member of the G protein-coupled pyrimidinergic P2 receptor family that responds specifically to the extracellular nucleotide uridine diphosphate (UDP). Recently, the hP2Y6 receptor has been reported to mediate monocyte IL-8 production in response to UDP or lipopolysaccharide (LPS), but the role of hP2Y6 in regulating other pro-inflammatory cytokines or mediators is largely unknown. We demonstrate here that UDP specifically induces soluble TNF-alpha and IL-8 production in a promonocytic U937 cell line stably transfected with hP2Y6. However, we did not detect IL-1alpha, IL-1beta, IL-6, IL-10, IL-18, and PGE2 in the conditioned media from the same cell line. These results distinguish UDP/P2Y6 signaling from LPS signaling. Interestingly, UDP induces the production of IL-8, but not TNF-alpha, in human astrocytoma 1321N1 cell lines stably transfected with hP2Y6. Therefore, the immune effect of UDP/P2Y6 signaling on the production of proinflammatory cytokines is selective and dependent on cell types. We further identify that UDP can also induce the production of proinflammatory chemokines MCP-1 and IP-10 in hP2Y6 transfected promonocytic U937 cell lines, but not astrocytoma 1321N1 cell lines stably transfected with hP2Y6. From the Taqman analysis, UDP stimulation significantly upregulates the mRNA levels of IL-8, IP-10, and IL-1beta, but not TNF-alpha. Taken together, these new findings expand the pro-inflammatory biology of UDP mediated by the P2Y6 receptor.

Inhibition of intimal hyperplasia in transgenic mice conditionally expressing the chemokine-binding protein M3.

Chemokines have been implicated in the pathogenesis of a wide variety of diseases. This report describes the generation of transgenic mice that conditionally express M3, a herpesvirus protein that binds and inhibits chemokines. In response to doxycycline, M3 expression was induced in a variety of tissues and M3 was detectable in the blood by Western blotting. No gross or histological abnormalities were seen in mice expressing M3. To determine whether M3 expression could modify a significant pathophysiological response, we examined its effect on the development of intimal hyperplasia in response to femoral arterial injury. Intimal hyperplasia is thought to play a critical role in the development of restenosis after percutaneous transluminal coronary angioplasty and in the progression of atherosclerosis. Induction of M3 expression resulted in a 67% reduction in intimal area and a 68% reduction in intimal/medial ratio after femoral artery injury. These data support a role for chemokines in regulating intimal hyperplasia and suggest that M3 may be effective in attenuating this process. This transgenic mouse model should be a valuable tool for investigating the role of chemokines in a variety of pathological states.

Cloning and pharmacological characterization of CXCR1 and CXCR2 from Macaca fascicularis.

Two genes with high sequence homology to human CXCR1 (hCXCR1) and CXCR2 (hCXCR2) were cloned from blood of cynomolgus monkey (Macaca fascicularis). Comparison of the expression pattern of these receptors in different species demonstrated that, like in humans, cynomolgus CXCR1 (cCXCR1) and CXCR2 (cCXCR2) are highly expressed in blood. Membranes from transfected BaF3 cells expressing cCXCR1 bind interleukin (IL)-8 with an affinity similar to hCXCR1 (Kd values, 170 +/- 87 and 103 +/- 37 pM, respectively) and show low binding affinity to Gro-alpha. Cynomolgus CXCR2 also binds hIL-8 but with somewhat higher affinity than the hCXCR2 (46 +/- 28 and 220 +/- 14 pM, respectively). Surprisingly, cCXCR2 has a reduced binding affinity to hGro-alpha (3.7 +/- 2.2 nM), a specific ligand of hCXCR2 (540 +/- 140 pM). Furthermore, the CXCR2-specific antagonist SB225002 [N-(2-hydroxy-4-nitrophenyl)-N'-(2-bromophenyl)urea] is 10-fold more potent in inhibiting IL-8 binding to hCXCR2 than to cCXCR2, suggesting that some of the observed differences in the amino acid sequences of the human and monkey receptor affect ligand binding sites or the conformation of the receptor. Both cynomolgus receptors were functionally active in inducing guanosine 5'-O-(3-thio)triphosphate exchange on membranes in response to IL-8 and Gro-alpha and in mediating chemotactic activity of recombinant BA/F3 cells in response to IL-8 and Gro-alpha. These results identify the products of the novel cynomolgus genes as functional homologs of hCXCR1 and hCXCR2.

Conditional expression of murine Flt3 ligand leads to expansion of multiple dendritic cell subsets in peripheral blood and tissues of transgenic mice.

The analysis of the development and function of distinct subsets of murine dendritic cells (DC) has been hampered by the limited number of these cells in vivo. To circumvent this limitation we have developed a conditional transgenic mouse model for producing large numbers of DC. We used the tetracycline-inducible system to conditionally express murine Flt3 ligand (FL), a potent hemopoietic growth factor that promotes the differentiation and mobilization of DC. Acute treatment (96 h) of the transgenic animals with the tetracycline analog doxycycline (DOX) promoted an approximately 200-fold increase in serum levels of FL without affecting the number of circulating DC. However, within 1 wk of DOX treatment, the relative number of DC in peripheral blood increased from approximately 8 to approximately 40%. Interestingly, both the levels of FL and the number of DC remained elevated for at least 9 mo with continual DOX treatment. Chronic treatment of the mice with DOX led to dramatic increases in the number of DC in multiple tissues without any apparent pathological consequences. Most DC populations were expanded, including immature and mature DC, myeloid (CD11c(+)CD11b(+)CD8a(-)), lymphoid (CD11c(+)CD11b(-)CD8a(+)), and the recently defined plasmacytoid (pDC) subsets. Finally, transplantation of BM from green fluorescent protein-expressing mice into lethally irradiated transgenic mice followed by subsequent DOX treatment led to expansion of green fluorescent protein-labeled DC. The transgenic mice described here should thus provide a readily available source of multiple DC subsets and should facilitate the analysis of their role in homeostasis and disease.

Interleukin-23 rather than interleukin-12 is the critical cytokine for autoimmune inflammation of the brain.

Interleukin-12 (IL-12) is a heterodimeric molecule composed of p35 and p40 subunits. Analyses in vitro have defined IL-12 as an important factor for the differentiation of naive T cells into T-helper type 1 CD4+ lymphocytes secreting interferon-gamma (refs 1, 2). Similarly, numerous studies have concluded that IL-12 is essential for T-cell-dependent immune and inflammatory responses in vivo, primarily through the use of IL-12 p40 gene-targeted mice and neutralizing antibodies against p40. The cytokine IL-23, which comprises the p40 subunit of IL-12 but a different p19 subunit, is produced predominantly by macrophages and dendritic cells, and shows activity on memory T cells. Evidence from studies of IL-23 receptor expression and IL-23 overexpression in transgenic mice suggest, however, that IL-23 may also affect macrophage function directly. Here we show, by using gene-targeted mice lacking only IL-23 and cytokine replacement studies, that the perceived central role for IL-12 in autoimmune inflammation, specifically in the brain, has been misinterpreted and that IL-23, and not IL-12, is the critical factor in this response. In addition, we show that IL-23, unlike IL-12, acts more broadly as an end-stage effector cytokine through direct actions on macrophages.

Disruption of CCL21-induced chemotaxis in vitro and in vivo by M3, a chemokine-binding protein encoded by murine gammaherpesvirus 68.

Chemokine-binding proteins represent a novel class of antichemokine agents encoded by poxviruses and herpesviruses. One such protein is encoded by the M3 gene present in the murine gammaherpesvirus 68 (MHV-68) genome. The M3 gene encodes a secreted 44-kDa protein that binds with high affinity to certain murine and human chemokines and has been shown to block chemokine signaling in vitro. However, there has been no direct evidence that M3 blocks chemokine activity in vivo, nor has the nature of M3-chemokine interaction been defined. To better understand the ability of M3 to block chemokine activity in vivo, we examined its interaction with a specific subset of chemokines expressed in lymphoid tissues, areas where gammaherpesviruses characteristically establish latency. Here we show that M3 blocks in vitro chemotaxis induced by CCL19 and CCL21, chemokines expressed constitutively in secondary lymphoid tissues. Moreover, we provide evidence that chemokine M3 binding exhibits positive cooperativity. In vivo, the expression of M3 in the pancreas of transgenic mice inhibits recruitment of lymphocytes induced by transgenic expression of CCL21 in this organ. The ability of M3 to block the biological activity of chemokines may represent an important strategy used by MHV-68 to evade immune detection and favor viral replication in the infected host.

Interactions between hemopoietically derived TNF and central nervous system-resident glial chemokines underlie initiation of autoimmune inflammation in the brain.

Tumor necrosis factor is a proinflammatory cytokine that induces directly many of the components required for inflammation to proceed rapidly. We show in this study that the interplay between TNF and chemokines, now recognized to be essential for normal secondary lymphoid tissue development, is also a feature of CNS inflammation, and that the two apparently dissimilar biological processes share many properties. Thus, induction of seven chemokines, including T cell activation gene 3 (TCA3), monocyte chemoattractant protein-1, and IFN-gamma-inducible protein-10 within the CNS during experimental autoimmune encephalomyelitis fails to occur early in the inflammatory process in TNF-deficient mice, despite local expression of monokines and IFN-gamma. The critical source of TNF in CNS inflammation is the infiltrating hemopoietic cell, and, in its absence, chemokine expression by irradiation-resistant CNS-resident cells fails. The CCR8 ligand, TCA3, is shown to be produced predominantly by resident microglia of the CNS in response to TNF. Using CCR8(-/-) mice, evidence is provided that TCA3-CCR8 interactions contribute to rapid-onset CNS inflammation. Thus, through TNF production, the hemopoietic compartment initiates the signals for its own movement into tissues, although the tissue ultimately defines the nature of that movement. Chemokines are a major, although not exclusive, mechanism by which tissues regulate leukocyte movement in response to TNF.

Transient lung-specific expression of the chemokine KC improves outcome in invasive aspergillosis.

Invasive aspergillosis is a common and devastating pneumonia in immunocompromised hosts. Neutrophils are critical for defense against this infection, and ELR+ CXC chemokines are potent neutrophil chemoattractants. We hypothesized that transient lung-specific overexpression of one such ligand, KC, in mice with invasive aspergillosis improves the outcome of disease. We generated mice in which transgenic expression of KC was limited to the lungs and occurred only upon exposure to tetracycline analogues, and we exposed them to doxycycline after the onset of invasive aspergillosis. Transgenic mice had a threefold greater survival, a 74% lower lung fungal burden, a greater magnitude of lung KC induction, and an earlier and higher peak of lung neutrophil influx compared with wild-type mice. In addition to a higher number of neutrophils, we found a 1.8-fold higher number of monocytes-macrophages in the lungs of transgenic mice as compared with wild-type mice. Furthermore, transgenic mice had greater lung expression of interferon-gamma and interleukin-12 in response to infection, suggesting that transgenic expression of KC indirectly regulated the expression of other cytokines associated with improved host defense against this pathogen. Taken together, these data suggest that overexpression of KC in the lung in the setting of established invasive aspergillosis results in improved host defense and outcome of disease.

Increased responsiveness of murine eosinophils to MIP-1beta (CCL4) and TCA-3 (CCL1) is mediated by their specific receptors, CCR5 and CCR8.

In the present study, we investigated the regulation of chemokine-mediated responses and receptor expression on eosinophils from mice. MIP-1alpha (CCL3) and eotaxin (CCL11) induced a significant and only partially overlapping intracellular calcium flux in antigen-elicited and peripheral blood eosinophils, and MCP-1 (CCL2), MDC (CCL22), MIP-1beta (CCL4), and TCA-3 (CCL1) did not. To demonstrate functional use of the specific receptors, we examined chemotactic responses. Peripheral blood eosinophils migrated toward MIP-1alpha (CCL3) and eotaxin (CCL11) but not MCP-1 (CCL2), MDC (CCL22), MIP-1beta (CCL4), and TCA-3 (CCL1). Antigen-elicited eosinophils migrated toward MIP-1alpha (CCL3) and eotaxin (CCL11), but also migrated in response to MIP-1beta (CCL4) and TCA-3 (CCL1), suggesting the up-regulation of additional chemokine receptors on antigen-elicited eosinophils. The up-regulation of the additional chemokine-receptor responses appeared to be in part because of cytokine activation, because TNF-alpha and/or IL-4 were able to up-regulate CCR1, -3, -5, and -8 mRNA expression in eosinophils as well as migration responses to the appropriate ligands. Using antibodies specific for CCR5 and CCR8, the chemotactic response to MIP-1beta and TCA-3, respectively, was reduced significantly. Finally, the expression of these new receptors appears to have an effect on activation and degranulation because MIP-1beta (CCL4) and TCA-3 (CCL1) induce significant levels of LTC4 from elicited eosinophils. These results suggest that eosinophils may up-regulate and use additional chemokine receptors during progression of inflammatory, allergic responses for migration and activation.

Ectopic expression of the murine chemokines CCL21a and CCL21b induces the formation of lymph node-like structures in pancreas, but not skin, of transgenic mice.

The CC chemokine CCL21 is a potent chemoattractant for lymphocytes and dendritic cells in vitro. In the murine genome there are multiple copies of CCL21 encoding two CCL21 proteins that differ from each other by one amino acid at position 65 (either a serine or leucine residue). In this report, we examine the expression pattern and biological activities of both forms of CCL21. We found that although both serine and leucine forms are expressed in most tissues examined, the former was the predominant form in lymphoid organs while the latter was predominantly expressed in nonlymphoid organs. When expressed in transgenic pancreas, both forms of CCL21 were capable of inducing the formation of lymph node-like structures composed primarily of T and B cells and a few dendritic cells. Induction of lymph node-like structures by these CCL21 proteins, however, could not be reproduced in every tissue. For instance, no lymphocyte recruitment or accumulation was observed when CCL21 was overexpressed in the skin. We conclude that both forms of CCL21 protein are biologically equivalent in promoting lymphocyte recruitment to the pancreas, and that their ability to induce the formation of lymph node-like structures is dependent on the tissues in which they are expressed.

Central nervous system inflammation and neurological disease in transgenic mice expressing the CC chemokine CCL21 in oligodendrocytes.

To study the biological role of the chemokine ligands CCL19 and CCL21, we generated transgenic mice expressing either gene in oligodendrocytes of the CNS. While all transgenic mice expressing CCL19 in the CNS developed normally, most (18 of 26) of the CCL21 founder mice developed a neurological disease that was characterized by loss of landing reflex, tremor, and ataxia. These neurological signs were observed as early as postnatal day 9 and were associated with weight loss and death during the first 4 wk of life. Microscopic examination of the brain and spinal cord of CCL21 transgenic mice revealed scattered leukocytic infiltrates that consisted primarily of neutrophils and eosinophils. Additional findings included hypomyelination, spongiform myelinopathy with evidence of myelin breakdown, and reactive gliosis. Thus, ectopic expression of the CC chemokine CCL21, but not CCL19, induced a significant inflammatory response in the CNS. However, neither chemokine was sufficient to recruit lymphocytes into the CNS. These observations are in striking contrast to the reported activities of these molecules in vitro and may indicate specific requirements for their biological activity in vivo.