CXCR2 inhibition enables NASH-HCC immunotherapy.

OBJECTIVE: Hepatocellular carcinoma (HCC) is increasingly associated with non-alcoholic steatohepatitis (NASH). HCC immunotherapy offers great promise; however, recent data suggests NASH-HCC may be less sensitive to conventional immune checkpoint inhibition (ICI). We hypothesised that targeting neutrophils using a CXCR2 small molecule inhibitor may sensitise NASH-HCC to ICI therapy. DESIGN: Neutrophil infiltration was characterised in human HCC and mouse models of HCC. Late-stage intervention with anti-PD1 and/or a CXCR2 inhibitor was performed in murine models of NASH-HCC. The tumour immune microenvironment was characterised by imaging mass cytometry, RNA-seq and flow cytometry. RESULTS: Neutrophils expressing CXCR2, a receptor crucial to neutrophil recruitment in acute-injury, are highly represented in human NASH-HCC. In models of NASH-HCC lacking response to ICI, the combination of a CXCR2 antagonist with anti-PD1 suppressed tumour burden and extended survival. Combination therapy increased intratumoural XCR1+ dendritic cell activation and CD8+ T cell numbers which are associated with anti-tumoural immunity, this was confirmed by loss of therapeutic effect on genetic impairment of myeloid cell recruitment, neutralisation of the XCR1-ligand XCL1 or depletion of CD8+ T cells. Therapeutic benefit was accompanied by an unexpected increase in tumour-associated neutrophils (TANs) which switched from a protumour to anti-tumour progenitor-like neutrophil phenotype. Reprogrammed TANs were found in direct contact with CD8+ T cells in clusters that were enriched for the cytotoxic anti-tumoural protease granzyme B. Neutrophil reprogramming was not observed in the circulation indicative of the combination therapy selectively influencing TANs. CONCLUSION: CXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC.

Cancer-associated fibroblasts produce matrix-bound vesicles that influence endothelial cell function.

Intercellular communication between different cell types in solid tumors contributes to tumor growth and metastatic dissemination. The secretome of cancer-associated fibroblasts (CAFs) plays major roles in these processes. Using human mammary CAFs, we showed that CAFs with a myofibroblast phenotype released extracellular vesicles that transferred proteins to endothelial cells (ECs) that affected their interaction with immune cells. Mass spectrometry-based proteomics identified proteins transferred from CAFs to ECs, which included plasma membrane receptors. Using THY1 as an example of a transferred plasma membrane-bound protein, we showed that CAF-derived proteins increased the adhesion of a monocyte cell line to ECs. CAFs produced high amounts of matrix-bound EVs, which were the primary vehicles of protein transfer. Hence, our work paves the way for future studies that investigate how CAF-derived matrix-bound EVs influence tumor pathology by regulating the function of neighboring cancer, stromal, and immune cells.

Hapalosiphonacean cyanobacteria (Nostocales) thrived amid emerging embryophytes in an early Devonian (407-million-year-old) landscape.

Cyanobacteria have a long evolutionary history, well documented in marine rocks. They are also abundant and diverse in terrestrial environments; however, although phylogenies suggest that the group colonized land early in its history, paleontological documentation of this remains limited. The Rhynie chert (407 Ma), our best preserved record of early terrestrial ecosystems, provides an opportunity to illuminate aspects of cyanobacterial diversity and ecology as plants began to radiate across the land surface. We used light microscopy and super-resolution confocal laser scanning microscopy to study a new population of Rhynie cyanobacteria; we also reinvestigated previously described specimens that resemble the new fossils. Our study demonstrates that all are part of a single fossil species belonging to the Hapalosiphonaceae (Nostocales). Along with other Rhynie microfossils, these remains show that the accommodation of morphologically complex cyanobacteria to terrestrial ecosystems transformed by embryophytes was well underway more than 400 million years ago.

The efficacy of the benzimidazoles oxfendazole and flubendazole against Litomosoides sigmodontis is dependent on the adaptive and innate immune system.

Filarial nematodes can cause debilitating diseases such as lymphatic filariasis and onchocerciasis. Oxfendazole (OXF) is one promising macrofilaricidal candidate with improved oral availability compared to flubendazole (FBZ), and OXF is currently under preparation for phase 2 clinical trials in filariasis patients. This study aimed to investigate the immune system's role during treatment with OXF and FBZ and explore the potential to boost the treatment efficacy via stimulation of the immune system. Wild type (WT) BALB/c, eosinophil-deficient ΔdblGata1, IL-4r/IL-5-/-, antibody-deficient µMT and B-, T-, NK-cell and ILC-deficient Rag2/IL-2rγ-/- mice were infected with the rodent filaria Litomosoides sigmodontis and treated with an optimal and suboptimal regimen of OXF and FBZ for up to 5 days. In the second part, WT mice were treated for 2-3 days with a combination of OXF and IL-4, IL-5, or IL-33. Treatment of WT mice reduced the adult worm burden by up to 94% (OXF) and 100% (FBZ) compared to vehicle controls. In contrast, treatment efficacy was lower in all immunodeficient strains with a reduction of up to 90% (OXF) and 75% (FBZ) for ΔdblGata1, 50 and 92% for IL-4r/IL-5-/-, 64 and 78% for µMT or 0% for Rag2/IL-2rγ-/- mice. The effect of OXF on microfilariae and embryogenesis displayed a similar pattern, while FBZ's ability to prevent microfilaremia was independent of the host's immune status. Furthermore, flow cytometric analysis revealed strain-and treatment-specific immunological changes. The efficacy of a shortened 3-day treatment of OXF (-33% adult worms vs. vehicle) could be boosted to a 91% worm burden reduction via combination with IL-5, but not IL-4 or IL-33. Our results suggest that various components of the immune system support the filaricidal effect of benzimidazoles in vivo and present an opportunity to boost treatment efficacy.

Lung extracellular matrix modulates KRT5+ basal cell activity in pulmonary fibrosis.

Aberrant expansion of KRT5+ basal cells in the distal lung accompanies progressive alveolar epithelial cell loss and tissue remodelling during fibrogenesis in idiopathic pulmonary fibrosis (IPF). The mechanisms determining activity of KRT5+ cells in IPF have not been delineated. Here, we reveal a potential mechanism by which KRT5+ cells migrate within the fibrotic lung, navigating regional differences in collagen topography. In vitro, KRT5+ cell migratory characteristics and expression of remodelling genes are modulated by extracellular matrix (ECM) composition and organisation. Mass spectrometry- based proteomics revealed compositional differences in ECM components secreted by primary human lung fibroblasts (HLF) from IPF patients compared to controls. Over-expression of ECM glycoprotein, Secreted Protein Acidic and Cysteine Rich (SPARC) in the IPF HLF matrix restricts KRT5+ cell migration in vitro. Together, our findings demonstrate how changes to the ECM in IPF directly influence KRT5+ cell behaviour and function contributing to remodelling events in the fibrotic niche.

Unbalanced Arginine pathway and altered maturation of pleural macrophages in Th2-deficient mice during Litomosoides sigmodontis filarial infection.

Filarial parasites are tissue dwelling worms transmitted by hematophagous vectors. Understanding the mechanisms regulating microfilariae (the parasite offspring) development is a prerequisite for controlling transmission in filarial infections. Th2 immune responses are key for building efficient anti-parasite responses but have been shown to also lead to detrimental tissue damage in the presence of microfilariae. Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity was therefore used to characterize pleuropulmonary pathology and associated immune responses in wild-type and Th2 deficient mice. Wild-type and Th2-deficient mice (Il-4rα-/-/Il-5-/- ) were infected with L. sigmodontis and parasite outcome was analyzed during the patent phase (when microfilariae are in the general circulation). Pleuropulmonary manifestations were investigated and pleural and bronchoalveolar cells were characterized by RNA analysis, imaging and/or flow cytometry focusing on macrophages. Il-4rα-/-/Il-5-/- mice were hypermicrofilaremic and showed an enhanced filarial survival but also displayed a drastic reduction of microfilaria-driven pleural cavity pathologies. In parallel, pleural macrophages from Il-4rα-/-/Il-5-/- mice lacked expression of prototypical alternative activation markers RELMα and Chil3 and showed an altered balance of some markers of the arginine metabolic pathway. In addition, monocytes-derived F4/80intermediate macrophages from infected Il-4rα-/-/Il-5-/- mice failed to mature into resident F4/80high large macrophages. Altogether these data emphasize that the presence of both microfilariae and IL-4R/IL-5 signaling are critical in the development of the pathology and in the phenotype of macrophages. In Il-4rα-/-/Il-5-/- mice, the balance is in favor of parasite development while limiting the pathology associated with the host immune response.

A noninvasive iRFP713 p53 reporter reveals dynamic p53 activity in response to irradiation and liver regeneration in vivo.

Genetically encoded probes are widely used to visualize cellular processes in vitro and in vivo. Although effective in cultured cells, fluorescent protein tags and reporters are suboptimal in vivo because of poor tissue penetration and high background signal. Luciferase reporters offer improved signal-to-noise ratios but require injections of luciferin that can lead to variable responses and that limit the number and timing of data points that can be gathered. Such issues in studying the critical transcription factor p53 have limited insight on its activity in vivo during development and tissue injury responses. Here, by linking the expression of the near-infrared fluorescent protein iRFP713 to a synthetic p53-responsive promoter, we generated a knock-in reporter mouse that enabled noninvasive, longitudinal analysis of p53 activity in vivo in response to various stimuli. In the developing embryo, this model revealed the timing and localization of p53 activation. In adult mice, the model monitored p53 activation in response to irradiation and paracetamol- or CCl4-induced liver regeneration. After irradiation, we observed potent and sustained activation of p53 in the liver, which limited the production of reactive oxygen species (ROS) and promoted DNA damage resolution. We propose that this new reporter may be used to further advance our understanding of various physiological and pathophysiological p53 responses.

Neutrophil dynamics in the tumor microenvironment.

The tumor microenvironment profoundly influences the behavior of recruited leukocytes and tissue-resident immune cells. These immune cells, which inherently have environmentally driven plasticity necessary for their roles in tissue homeostasis, dynamically interact with tumor cells and the tumor stroma and play critical roles in determining the course of disease. Among these immune cells, neutrophils were once considered much more static within the tumor microenvironment; however, some of these earlier assumptions were the product of the notorious difficulty in manipulating neutrophils in vitro. Technological advances that allow us to study neutrophils in context are now revealing the true roles of neutrophils in the tumor microenvironment. Here we discuss recent data generated by some of these tools and how these data might be synthesized into more elegant ways of targeting these powerful and abundant effector immune cells in the clinic.

Microfilariae Trigger Eosinophil Extracellular DNA Traps in a Dectin-1-Dependent Manner.

Eosinophils mediate protection against filarial nematodes. Our results demonstrate that eosinophil extracellular traps (EETosis) are induced by microfilariae and infective L3 larvae of Litomosoides sigmodontis. These extracellular DNA traps inhibit microfilariae motility in a DNA- and contact-dependent manner in vitro. Accordingly, microfilariae-injection triggers DNA release in an eosinophil-dependent manner in vivo and microfilariae covered with DNA traps are cleared more rapidly. Using dectin-1, we identify the required receptor for the microfilariae-induced EETosis, whereas signaling via other C-type lectin receptors, prior priming of eosinophils, and presence of antibodies are not required. The DNA released upon microfilariae-induced EETosis is mainly of mitochondrial origin, but acetylated and citrullinated histones are found within the traps. We further demonstrate that the presented DNA-dependent inhibition of microfilariae motility by eosinophils represents a conserved mechanism, as microfilariae from L. sigmodontis and the canine heartworm Dirofilaria immitis induce ETosis in murine and human eosinophils.

The transcription factor EGR2 is indispensable for tissue-specific imprinting of alveolar macrophages in health and tissue repair.

Alveolar macrophages are the most abundant macrophages in the healthy lung where they play key roles in homeostasis and immune surveillance against airborne pathogens. Tissue-specific differentiation and survival of alveolar macrophages rely on niche-derived factors, such as granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor­ß (TGF-ß). However, the nature of the downstream molecular pathways that regulate the identity and function of alveolar macrophages and their response to injury remain poorly understood. Here, we identify that the transcription factor EGR2 is an evolutionarily conserved feature of lung alveolar macrophages and show that cell-intrinsic EGR2 is indispensable for the tissue-specific identity of alveolar macrophages. Mechanistically, we show that EGR2 is driven by TGF-ß and GM-CSF in a PPAR-γ­dependent manner to control alveolar macrophage differentiation. Functionally, EGR2 was dispensable for the regulation of lipids in the airways but crucial for the effective handling of the respiratory pathogen Streptococcus pneumoniae. Last, we show that EGR2 is required for repopulation of the alveolar niche after sterile, bleomycin-induced lung injury and demonstrate that EGR2-dependent, monocyte-derived alveolar macrophages are vital for effective tissue repair after injury. Collectively, we demonstrate that EGR2 is an indispensable component of the transcriptional network controlling the identity and function of alveolar macrophages in health and disease.

Microfilaria-dependent thoracic pathology associated with eosinophilic and fibrotic polyps in filaria-infected rodents.

BACKGROUND: Pulmonary manifestations are regularly reported in both human and animal filariasis. In human filariasis, the main known lung manifestations are the tropical pulmonary eosinophilia syndrome. Its duration and severity are correlated with the presence of microfilariae. Litomosoides sigmodontis is a filarial parasite residing in the pleural cavity of rodents. This model is widely used to understand the immune mechanisms that are established during infection and for the screening of therapeutic molecules. Some pulmonary manifestations during the patent phase of infection with L. sigmodontis have been described in different rodent hosts more or less permissive to infection. METHODS: Here, the permissive Mongolian gerbil (Meriones unguiculatus) was infected with L. sigmodontis. Prevalence and density of microfilariae and adult parasites were evaluated. Lungs were analyzed for pathological signatures using immunohistochemistry and 3D imaging techniques (two-photon and light sheet microscopy). RESULTS: Microfilaremia in gerbils was correlated with parasite load, as amicrofilaremic individuals had fewer parasites in their pleural cavities. Fibrotic polypoid structures were observed on both pleurae of infected gerbils. Polyps were of variable size and developed from the visceral mesothelium over the entire pleura. The larger polyps were vascularized and strongly infiltrated by immune cells such as eosinophils, macrophages or lymphocytes. The formation of these structures was induced by the presence of adult filariae since small and rare polyps were observed before patency, but they were exacerbated by the presence of gravid females and microfilariae. CONCLUSIONS: Altogether, these data emphasize the role of host-specific factors in the pathogenesis of filarial infections.

S100A8/S100A9 deficiency increases neutrophil activation and protective immune responses against invading infective L3 larvae of the filarial nematode Litomosoides sigmodontis.

Neutrophils are essentially involved in protective immune responses against invading infective larvae of filarial nematodes. The present study investigated the impact of S100A8/S100A9 on protective immune responses against the rodent filarial nematode Litomosoides sigmodontis. S100A9 forms with S100A8 the heterodimer calprotectin, which is expressed by circulating neutrophils and monocytes and mitigates or amplifies tissue damage as well as inflammation depending on the immune environment. Mice deficient for S100A8/A9 had a significantly reduced worm burden in comparison to wildtype (WT) animals 12 days after infection (dpi) with infective L3 larvae, either by the vector or subcutaneous inoculation, the latter suggesting that circumventing natural immune responses within the epidermis and dermis do not alter the phenotype. Nevertheless, upon intradermal injection of L3 larvae, increased total numbers of neutrophils, eosinophils and macrophages were observed within the skin of S100A8/A9-/- mice. Furthermore, upon infection the bronchoalveolar and thoracic cavity lavage of S100A8/A9-/- mice showed increased concentrations of CXCL-1, CXCL-2, CXCL-5, as well as elastase in comparison to the WT controls. Neutrophils from S100A8/A9-/- mice exhibited an increased in vitro activation and reduced L3 larval motility more effectively in vitro compared to WT neutrophils. The depletion of neutrophils from S100A8/A9-/- mice prior to L. sigmodontis infection until 5dpi abrogated the protective effect and led to an increased worm burden, indicating that neutrophils mediate enhanced protective immune responses against invading L3 larvae in S100A8/A9-/- mice. Interestingly, complete circumvention of protective immune responses in the skin and the lymphatics by intravenous injection of L3 larvae reversed the phenotype and resulted in an increased worm burden in S100A8/A9-/- mice. In summary, our results reveal that lack of S100A8/S100A9 triggers L3-induced inflammatory responses, increasing chemokine levels, granulocyte recruitment as well as neutrophil activation and therefore impairs larval migration and susceptibility for filarial infection.

IL-4 receptor dependent expansion of lung CD169+ macrophages in microfilaria-driven inflammation.

Lung disease is regularly reported in human filarial infections but the molecular pathogenesis of pulmonary filariasis is poorly understood. We used Litomosoides sigmodontis, a rodent filaria residing in the pleural cavity responsible for pleural inflammation, to model responses to human filarial infections and probe the mechanisms. Wild-type and Th2-deficient mice (ΔdblGata1 and Il-4receptor(r)a-/-/IL-5-/-) were infected with L. sigmodontis. Survival and growth of adult filariae and prevalence and density of microfilariae were evaluated. Cells and cytokines in the pleural cavity and bronchoalveolar space were characterized by imaging, flow cytometry and ELISA. Inflammatory pathways were evaluated by transcriptomic microarrays and lungs were isolated and analyzed for histopathological signatures. 40% of WT mice were amicrofilaremic whereas almost all mutant mice display blood microfilaremia. Microfilariae induced pleural, bronchoalveolar and lung-tissue inflammation associated with an increase in bronchoalveolar eosinophils and perivascular macrophages, production of mucus, visceral pleura alterations and fibrosis. Inflammation and pathology were decreased in Th2-deficient mice. An IL-4R-dependent increase of CD169 was observed on pleural and bronchoalveolar macrophages in microfilaremic mice. CD169+ tissue-resident macrophages were identified in the lungs with specific localizations. Strikingly, CD169+ macrophages increased significantly in the perivascular area in microfilaremic mice. These data describe lung inflammation and pathology in chronic filariasis and emphasize the role of Th2 responses according to the presence of microfilariae. It is also the first report implicating CD169+ lung macrophages in response to a Nematode infection.

Migratory phase of Litomosoides sigmodontis filarial infective larvae is associated with pathology and transient increase of S100A9 expressing neutrophils in the lung.

Filarial infections are tropical diseases caused by nematodes of the Onchocercidae family such as Mansonella perstans. The infective larvae (L3) are transmitted into the skin of vertebrate hosts by blood-feeding vectors. Many filarial species settle in the serous cavities including M. perstans in humans and L. sigmodontis, a well-established model of filariasis in mice. L. sigmodontis L3 migrate to the pleural cavity where they moult into L4 around day 9 and into male and female adult worms around day 30. Little is known of the early phase of the parasite life cycle, after the L3 is inoculated in the dermis by the vector and enters the afferent lymphatic vessels and before the moulting processes in the pleural cavity. Here we reveal a pulmonary phase associated with lung damage characterized by haemorrhages and granulomas suggesting L3 reach the lung via pulmonary capillaries and damage the endothelium and parenchyma by crossing them to enter the pleural cavity. This study also provides evidence for a transient inflammation in the lung characterized by a very early recruitment of neutrophils associated with high expression levels of S100A8 and S100A9 proteins.

Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae.

Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4(+/1013)). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4(+/1013) mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4(+/1013) mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis.