Inhibition of P21-activated kinases 1 and 4 synergistically suppresses the growth of pancreatic cancer by stimulating anti-tumour immunity.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal types of cancer, and KRAS oncogene occurs in over 90% of cases. P21-activated kinases (PAK), containing six members (PAK1 to 6), function downstream of KRAS. PAK1 and PAK4 play important roles in carcinogenesis, but their combinational effect remains unknown. In this study, we have determined the effect of dual inhibition of PAK1 and PAK4 in PDA progression using knockout (KO) cancer cell lines. METHODS: Murine wild-type (WT) and PAK1KO pancreatic cancer cell lines were isolated from PAK1+/+ and PAK1-/- KPC (LSL-KrasG12D/+; LSL-Trp53 R172H/+; Pdx-1-Cre) mice. KPC PAK4KO and KPC PAK1&4 KO cell lines were generated from KPC WT and KPC PAK1KO cell lines respectively using the CRISPR-CAS9 gene knockout technique. PAK WT and KO cell lines were used in mouse models of pancreatic tumours. Cells and tumour tissue were also used in flow cytometry and proteomic studies. A human PDA tissue microarray was stained by immunohistochemistry. RESULTS: Double knock out of PAK1 and PAK4 caused complete regression of tumour in a syngeneic mouse model. PAK4KO inhibited tumour growth by stimulating a rapid increase of cytotoxic CD8+ T cell infiltration. PAK1KO synergistically with PAK4KO increased cytotoxic CD8+ T cell infiltration and stimulated a sustained infiltration of CD8+ T cells at a later phase to overcome the immune evasion in the PAK4KO tumour. The human PDA tissue microarray study showed the important role of PAK1 and PAK4 in intra-tumoral T-cell function. CONCLUSION: Our results demonstrated that dual inhibition of PAK1 and PAK4 synergistically suppressed PDA progression by stimulating cytotoxic CD8 + T cell response.

Systemic inflammatory response syndrome triggered by blood-borne pathogens induces prolonged dendritic cell paralysis and immunosuppression.

Blood-borne pathogens can cause systemic inflammatory response syndrome (SIRS) followed by protracted, potentially lethal immunosuppression. The mechanisms responsible for impaired immunity post-SIRS remain unclear. We show that SIRS triggered by pathogen mimics or malaria infection leads to functional paralysis of conventional dendritic cells (cDCs). Paralysis affects several generations of cDCs and impairs immunity for 3-4 weeks. Paralyzed cDCs display distinct transcriptomic and phenotypic signatures and show impaired capacity to capture and present antigens in vivo. They also display altered cytokine production patterns upon stimulation. The paralysis program is not initiated in the bone marrow but during final cDC differentiation in peripheral tissues under the influence of local secondary signals that persist after resolution of SIRS. Vaccination with monoclonal antibodies that target cDC receptors or blockade of transforming growth factor ß partially overcomes paralysis and immunosuppression. This work provides insights into the mechanisms of paralysis and describes strategies to restore immunocompetence post-SIRS.

IL-36 antagonism blunts the proliferation and migration of oral squamous cell carcinoma cells.

IL-36 is known to mediate inflammation and fibrosis. Nevertheless, IL-36 signalling axis has also been implicated in cancer, although understanding of exact contribution of IL-36 to cancer progression is very limited, partly due to existence of multiple IL-36 ligands with agonistic and antagonistic function. Here we explored the role of IL-36 in oral squamous cell carcinoma (OSCC). Firstly, we analyzed expression of IL-36 ligands and receptor and found that the expression of IL-36γ was significantly higher in head and neck cancer (HNSCC) than that of normal tissues, and that the high expression of IL-36γ predicted poor clinical outcomes. Secondly, we investigated the direct effect of IL-36γ on OSCC cells and found that IL-36γ stimulated proliferation of OSCC cells with high expression of IL-36R expression. Interestingly, IL-36γ also promoted migration of OSCC cells with low to high IL-36R expression. Critically, both proliferation and migration of OSCC cells induced by IL-36γ were abrogated by anti-IL-36R mAb. Fittingly, RNA sequence analysis revealed that IL-36γ regulated genes involved in cell cycle and cell division. In summary, our results showed that IL-36γ can be a tumor-promoting factor, and targeting of IL-36R signalling may be a beneficial targeted therapy for patients with abnormal IL-36 signalling.

Identification and Characterization of CD8+ CD27+ CXCR3- T Cell Dysregulation and Progression-Associated Biomarkers in Systemic Lupus Erythematosus.

Systemic Lupus Erythematosus (SLE) etiopathogenesis highlights the contributions of overproduction of CD4+ T cells and loss of immune tolerance. However, the involvement of CD8+ T cells in SLE pathology and disease progression remains unclear. Here, the comprehensive immune cell dysregulation in total 263 clinical peripheral blood samples composed of active SLE (aSLE), remission SLE (rSLE) and healthy controls (HCs) is investigated via mass cytometry, flow cytometry and single-cell RNA sequencing. This is observed that CD8+ CD27+ CXCR3- T cells are increased in rSLE compare to aSLE. Meanwhile, the effector function of CD8+ CD27+ CXCR3- T cells are overactive in aSLE compare to HCs and rSLE, and are positively associated with clinical SLE activity. In addition, the response of peripheral blood mononuclear cells (PBMCs) is monitored to interleukin-2 stimulation in aSLE and rSLE to construct dynamic network biomarker (DNB) model. It is demonstrated that DNB score-related parameters can faithfully predict the remission of aSLE and the flares of rSLE. The abundance and functional dysregulation of CD8+ CD27+ CXCR3- T cells can be potential biomarkers for SLE prognosis and concomitant diagnosis. The DNB score with accurate prediction to SLE disease progression can provide clinical treatment suggestions especially for drug dosage determination.

Safety, tolerability, pharmacokinetics and efficacy of HB0017, a humanized monoclonal antibody that targets interleukin-17A, in healthy participants and patients with moderate-to-severe plaque psoriasis.

BACKGROUND: Several interleukin (IL)-17 inhibitors have been approved for the treatment of moderate-to-severe plaque psoriasis (PsO). There is still scope for the development of affordable treatments for PsO. OBJECTIVES: To assess, in a phase Ia study, the safety, tolerability and pharmacokinetics (PK) of HB0017, a humanized monoclonal antibody that targets IL-17A, in healthy participants and patients with moderate-to-severe plaque PsO; and, in a phase Ib study, to assess the efficacy of HB0017 in patients with moderate-to-severe plaque PsO. METHODS: The phase Ia study (NCT04505033) was a randomized double-blind placebo-controlled dose-escalation study in healthy participants. Each cohort of 10 volunteers was randomly assigned to receive either a single dose of HB0017 (50 mg, 150 mg, 300 mg or 450 mg) or the matching placebo at a ratio of 4 : 1. The phase Ib study (NCT05442788) was a randomized double-blind placebo-controlled dose-escalation study in enrolled patients with moderate-to-severe plaque PsO. Each cohort of 10 patients was randomly assigned to receive either multiple doses of HB0017 (150 mg, 300 mg or 450 mg) or the matching placebo at a ratio of 4 : 1. RESULTS: HB0017 demonstrated dose-proportional linear PK and was tolerated across the dose range assessed. In the phase Ia and Ib studies, participants in both the HB0017 and placebo groups experienced treatment-emergent adverse events (69% vs. 87%, 96% vs. 100%, respectively). HB0017 demonstrated clinically meaningful effects in patients with moderate-to-severe plaque PsO. PASI 75 [≥ 75% improvement in Psoriasis Area and Severity Index (PASI)], PASI 90 (≥ 90% improvement in PASI) and static Physician Global Assessment (sPGA) 0/1 (i.e. 'clear' or 'almost clear') responses were 100% for the HB0017 300-mg group, with maximal improvements (100% or near 100% reductions from baseline) in PASI score observed at week 12, while the duration of effect was evident up to week 20. There was no clinical response in any participant in the placebo group in the phase Ib study. CONCLUSIONS: Overall, HB0017 showed acceptable safety and tolerability in both healthy participants and patients with moderate-to-severe plaque PsO. An encouraging signal of efficacy with a longer half-life provides HB0017 with the potential to be added to the currently available range of biologics targeting IL-17A.

Iguratimod attenuated fibrosis in systemic sclerosis via targeting early growth response 1 expression.

BACKGROUND: The early growth response 1 (EGR1) is a central transcription factor involved in systemic sclerosis (SSc) pathogenesis. Iguratimod is a synthesized anti-rheumatic disease-modifying drug, which shows drastic inhibition to EGR1 expression in B cells. This study is aiming to investigate the anti-fibrotic effect of iguratimod in SSc. METHODS: EGR1 was detected by immunofluorescence staining real-time PCR or western blot. Iguratimod was applied in EGR1 overexpressed or knockdown human dermal fibroblast, bleomycin pre-treated mice, tight skin 1 mice, and SSc skin xenografts. RNA sequencing was performed in cultured fibroblast and xenografts to identify the iguratimod regulated genes. RESULTS: EGR1 overexpressed predominantly in non-immune cells of SSc patients. Iguratimod reduced EGR1 expression in fibroblasts and neutralized changes of EGR1 response genes regulated by TGFß. The extracellular matrix (ECM) production and activation of fibroblasts were attenuated by iguratimod while EGR1 overexpression reversed this effect of iguratimod. Iguratimod ameliorated the skin fibrosis induced by bleomycin and hypodermal fibrosis in TSK-1 mice. Decreasing in the collagen content as well as the density of EGR1 or TGFß positive fibroblasts of skin xenografts from naïve SSc patients was observed after local treatment of iguratimod. CONCLUSION: Targeting EGR1 expression is a probable underlying mechanism for the anti-fibrotic effect of iguratimod.

RIPK3 controls MAIT cell accumulation during development but not during infection.

Cell death mechanisms in T lymphocytes vary according to their developmental stage, cell subset and activation status. The cell death control mechanisms of mucosal-associated invariant T (MAIT) cells, a specialized T cell population, are largely unknown. Here we report that MAIT cells express key necroptotic machinery; receptor-interacting protein kinase 3 (RIPK3) and mixed lineage kinase domain-like (MLKL) protein, in abundance. Despite this, we discovered that the loss of RIPK3, but not necroptotic effector MLKL or apoptotic caspase-8, specifically increased MAIT cell abundance at steady-state in the thymus, spleen, liver and lungs, in a cell-intrinsic manner. In contrast, over the course of infection with Francisella tularensis, RIPK3 deficiency did not impact the magnitude of the expansion nor contraction of MAIT cell pools. These findings suggest that, distinct from conventional T cells, the accumulation of MAIT cells is restrained by RIPK3 signalling, likely prior to thymic egress, in a manner independent of canonical apoptotic and necroptotic cell death pathways.

CIS controls the functional polarization of GM-CSF-derived macrophages.

The cytokine granulocyte-macrophage-colony stimulating factor (GM-CSF) possesses the capacity to differentiate monocytes into macrophages (MØs) with opposing functions, namely, proinflammatory M1-like MØs and immunosuppressive M2-like MØs. Despite the importance of these opposing biological outcomes, the intrinsic mechanism that regulates the functional polarization of MØs under GM-CSF signaling remains elusive. Here, we showed that GM-CSF-induced MØ polarization resulted in the expression of cytokine-inducible SH2-containing protein (CIS) and that CIS deficiency skewed the differentiation of monocytes toward immunosuppressive M2-like MØs. CIS deficiency resulted in hyperactivation of the JAK-STAT5 signaling pathway, consequently promoting downregulation of the transcription factor Interferon Regulatory Factor 8 (IRF8). Loss- and gain-of-function approaches highlighted IRF8 as a critical regulator of the M1-like polarization program. In vivo, CIS deficiency induced the differentiation of M2-like macrophages, which promoted strong Th2 immune responses characterized by the development of severe experimental asthma. Collectively, our results reveal a CIS-modulated mechanism that clarifies the opposing actions of GM-CSF in MØ differentiation and uncovers the role of GM-CSF in controlling allergic inflammation.

Distinct Metabolism of Bone Marrow Adipocytes and their Role in Bone Metastasis.

Bone marrow adipocytes (BMAs) represent 10% of the total fat mass of the human body and serve as an energy reservoir for the skeletal niche. They function as an endocrine organ by actively secreting fatty acids, cytokines, and adipokines. The volume of BMAs increases along with age, osteoporosis and/or obesity. With the rapid development of multi-omic analysis and the advance in in vivo imaging technology, further distinct characteristics and functions of BMAs have been revealed. There is accumulating evidence that BMAs are metabolically, biologically and functionally unique from white, brown, beige and pink adipocytes. Bone metastatic disease is an uncurable complication in cancer patients, where primary cancer cells spread from their original site into the bone marrow. Recent publications have highlighted those BMAs could also serve as a rich lipid source of fatty acids that can be utilized by the cancer cells during bone metastasis, particularly for breast, prostate, lung, ovarian and pancreatic cancer as well as melanoma. In this review, we summarize the novel progressions in BMAs metabolism, especially with multi-omic analysis and in vivo imaging technology. We also update the metabolic role of BMAs in bone metastasis, and their potential new avenues for diagnosis and therapies against metastatic cancers.

Discordance in STING-Induced Activation and Cell Death Between Mouse and Human Dendritic Cell Populations.

Stimulator of Interferon Genes (STING) is a cytosolic sensor of cyclic dinucleotides (CDNs). The activation of dendritic cells (DC) via the STING pathway, and their subsequent production of type I interferon (IFN) is considered central to eradicating tumours in mouse models. However, this contribution of STING in preclinical murine studies has not translated into positive outcomes of STING agonists in phase I & II clinical trials. We therefore questioned whether a difference in human DC responses could be critical to the lack of STING agonist efficacy in human settings. This study sought to directly compare mouse and human plasmacytoid DCs and conventional DC subset responses upon STING activation. We found all mouse and human DC subsets were potently activated by STING stimulation. As expected, Type I IFNs were produced by both mouse and human plasmacytoid DCs. However, mouse and human plasmacytoid and conventional DCs all produced type III IFNs (i.e., IFN-λs) in response to STING activation. Of particular interest, all human DCs produced large amounts of IFN-λ1, not expressed in the mouse genome. Furthermore, we also found differential cell death responses upon STING activation, observing rapid ablation of mouse, but not human, plasmacytoid DCs. STING-induced cell death in murine plasmacytoid DCs occurred in a cell-intrinsic manner and involved intrinsic apoptosis. These data highlight discordance between STING IFN and cell death responses in mouse and human DCs and caution against extrapolating STING-mediated events in mouse models to equivalent human outcomes.

Structural and functional insights into a novel pre-clinical-stage antibody targeting IL-17A for treatment of autoimmune diseases.

The pro-inflammatory cytokine interleukin-17A (IL-17A) is a key driver of multiple inflammatory and immune disorders. Therapeutic antibodies targeting IL-17A have been proven effective in treating patients with these diseases; however, large variations in clinical outcomes have been observed with different antibodies. In this study, we developed HB0017, a novel monoclonal antibody that targets human IL-17A. HB0017 specifically and strongly bound to human, cynomolgus monkey, and mouse IL-17A at the physiological interface with the IL-17A receptor. In human and monkey cells, HB0017 potently antagonized the functions of IL-17A through competitive binding. HB0017 functioned equivalently to that of clinically approved antibodies in terms of therapeutic efficacy for inflammatory disorders and psoriasis in a mouse model. The results indicate that HB0017 may be an alternative biological therapy for treating patients with inflammation and autoimmune diseases.

A Novel Bispecific Antibody Targeting PD-L1 and VEGF With Combined Anti-Tumor Activities.

Therapeutic monoclonal antibodies (mAbs) blocking immune checkpoints have been mainly used as monotherapy. Recently, combination therapy targeting multiple immune checkpoints has recently been explored to increase anti-cancer efficacy. Particularly, a single molecule targeting more than one checkpoints has been investigated. As dual blocking of PD-1/PD-L1 and VEGF/VEGFR has demonstrated synergism in anti-tumor activities, we developed a novel bispecific antibody, termed HB0025, which is formed via fusing the domain 2 of vascular endothelial growth factor receptor 1 (VEGFR1D2) and anti-PD-L1 mAb by using mAb-Trap technology. HB0025 almost completely retains the binding affinities and the biological activities in-vitro when compared with the parent anti-PD-L1 mAb or VEGFR1D2 fusion protein. Preclinical data demonstrated that HB0025 was more effective in inhibiting cancer growth than anti PD-L1 mAb or VEGFR1D2 fusion protein. Thus, our bispecific antibody may bring about greater clinical benefits and broader indications.

Targeting the CCL2/CCR2 Axis in Cancer Immunotherapy: One Stone, Three Birds?

CCR2 is predominantly expressed by monocytes/macrophages with strong proinflammatory functions, prompting the development of CCR2 antagonists to dampen unwanted immune responses in inflammatory and autoimmune diseases. Paradoxically, CCR2-expressing monocytes/macrophages, particularly in tumor microenvironments, can be strongly immunosuppressive. Thus, targeting the recruitment of immunosuppressive monocytes/macrophages to tumors by CCR2 antagonism has recently been investigated as a strategy to modify the tumor microenvironment and enhance anti-tumor immunity. We present here that beneficial effects of CCR2 antagonism in the tumor setting extend beyond blocking chemotaxis of suppressive myeloid cells. Signaling within the CCL2/CCR2 axis shows underappreciated effects on myeloid cell survival and function polarization. Apart from myeloid cells, T cells are also known to express CCR2. Nevertheless, tissue homing of Treg cells among T cell populations is preferentially affected by CCR2 deficiency. Further, CCR2 signaling also directly enhances Treg functional potency. Thus, although Tregs are not the sole type of T cells expressing CCR2, the net outcome of CCR2 antagonism in T cells favors the anti-tumor arm of immune responses. Finally, the CCL2/CCR2 axis directly contributes to survival/growth and invasion/metastasis of many types of tumors bearing CCR2. Together, CCR2 links to two main types of suppressive immune cells by multiple mechanisms. Such a CCR2-assoicated immunosuppressive network is further entangled with paracrine and autocrine CCR2 signaling of tumor cells. Strategies to target CCL2/CCR2 axis as cancer therapy in the view of three types of CCR2-expessing cells in tumor microenvironment are discussed.

Differential requirement for the Polycomb repressor complex 2 in dendritic cell and tissue-resident myeloid cell homeostasis.

Dendritic cells (DCs) and macrophages are at the forefront of immune responses, modifying their transcriptional programs in response to their tissue environment or immunological challenge. Posttranslational modifications of histones, such as histone H3 lysine-27 trimethylation (H3K27me3) by the Polycomb repressive complex 2 (PRC2), are tightly associated with epigenetic regulation of gene expression. To explore whether H3K27me3 is involved in either the establishment or function of the mononuclear phagocyte system, we selectively deleted core components of PRC2, either EZH2 or SUZ12, in CD11c-expressing myeloid cells. Unexpectedly, EZH2 deficiency neither prevented the deposition and maintenance of H3K27me3 in DCs nor hindered DC/macrophage homeostasis. In contrast, SUZ12 deficiency markedly impaired the capacity of DCs and macrophages to maintain H3K27me3. SUZ12 ablation induced a rapid loss of the alveolar macrophage and Langerhans cell networks under both steady state and inflammatory conditions because these cells could no longer proliferate to facilitate their self-renewal. Despite the reduced H3K27me3, DC development and function were unaffected by SUZ12 ablation, suggesting that PRC2-mediated gene repression was dispensable for DC homeostasis. Thus, the role of SUZ12 highlights the fundamentally different homeostatic mechanisms used by tissue-resident myeloid cells versus DCs.

BCL-XL antagonism selectively reduces neutrophil life span within inflamed tissues without causing neutropenia.

Neutrophils help to clear pathogens and cellular debris, but can also cause collateral damage within inflamed tissues. Prolonged neutrophil residency within an inflammatory niche can exacerbate tissue pathology. Using both genetic and pharmacological approaches, we show that BCL-XL is required for the persistence of neutrophils within inflammatory sites in mice. We demonstrate that a selective BCL-XL inhibitor (A-1331852) has therapeutic potential by causing apoptosis in inflammatory human neutrophils ex vivo. Moreover, in murine models of acute and chronic inflammatory disease, it reduced inflammatory neutrophil numbers and ameliorated tissue pathology. In contrast, there was minimal effect on circulating neutrophils. Thus, we show a differential survival requirement in activated neutrophils for BCL-XL and reveal a new therapeutic approach to neutrophil-mediated diseases.

Type 1 conventional dendritic cell fate and function are controlled by DC-SCRIPT.

The functional diversification of dendritic cells (DCs) is a key step in establishing protective immune responses. Despite the importance of DC lineage diversity, its genetic basis is not fully understood. The transcription factor DC-SCRIPT is expressed in conventional DCs (cDCs) and their committed bone marrow progenitors but not in plasmacytoid DCs (pDCs). We show that mice lacking DC-SCRIPT displayed substantially impaired development of IRF8 (interferon regulatory factor 8)-dependent cDC1, whereas cDC2 numbers increased marginally. The residual DC-SCRIPT-deficient cDC1s had impaired capacity to capture and present cell-associated antigens and to secrete IL-12p40, two functional hallmarks of this population. Genome-wide mapping of DC-SCRIPT binding and gene expression analyses revealed a key role for DC-SCRIPT in maintaining cDC1 identity via the direct regulation of cDC1 signature genes, including Irf8 Our study reveals DC-SCRIPT to be a critical component of the gene regulatory program shaping the functional attributes of cDC1s.

TBK1 and IKKε Act Redundantly to Mediate STING-Induced NF-κB Responses in Myeloid Cells.

Stimulator of Interferon Genes (STING) is a critical component of host innate immune defense but can contribute to chronic autoimmune or autoinflammatory disease. Once activated, the cyclic guanosine monophosphate (GMP)-adenosine monophosphate (AMP) (cGAMP) synthase (cGAS)-STING pathway induces both type I interferon (IFN) expression and nuclear factor-κB (NF-κB)-mediated cytokine production. Currently, these two signaling arms are thought to be mediated by a single upstream kinase, TANK-binding kinase 1 (TBK1). Here, using genetic and pharmacological approaches, we show that TBK1 alone is dispensable for STING-induced NF-κB responses in human and mouse immune cells, as well as in vivo. We further demonstrate that TBK1 acts redundantly with IκB kinase ε (IKKε) to drive NF-κB upon STING activation. Interestingly, we show that activation of IFN regulatory factor 3 (IRF3) is highly dependent on TBK1 kinase activity, whereas NF-κB is significantly less sensitive to TBK1/IKKε kinase inhibition. Our work redefines signaling events downstream of cGAS-STING. Our findings further suggest that cGAS-STING will need to be targeted directly to effectively ameliorate the inflammation underpinning disorders associated with STING hyperactivity.

IL-17 production by tissue-resident MAIT cells is locally induced in children with pneumonia.

Community-acquired pneumonia (CAP) contributes substantially to morbidity and mortality in children under the age of 5 years. In examining bronchoalveolar lavages (BALs) of children with CAP, we found that interleukin-17 (IL-17) production was significantly increased in severe CAP. Immune profiling showed that mucosal-associated invariant T (MAIT) cells from the BALs, but not blood, of CAP patients actively produced IL-17 (MAIT17). Single-cell RNA-sequencing revealed that MAIT17 resided in a BAL-resident PLZFhiCD103+ MAIT subset with high expression of hypoxia-inducible factor 1α (HIF-1α), reflecting the hypoxic state of the inflamed tissue. CAP BALs also contained a T-bet+ MAIT1 subset and a novel DDIT3+ (DNA damage-inducible transcript 3-positive) MAIT subset with low expression of HIF1A. Furthermore, MAIT17 differed from T-helper type 17 (Th17) cells in the expression of genes related to tissue location, innateness, and cytotoxicity. Finally, we showed that BAL monocytes were hyper-inflammatory and elicited differentiation of MAIT17. Thus, tissue-resident MAIT17 cells are induced at the infected respiratory mucosa, likely influenced by inflammatory monocytes, and contribute to IL-17-mediated inflammation during CAP.