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.

ObjectiveHepatocellular 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.DesignNeutrophil 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.ResultsNeutrophils 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.ConclusionCXCR2-inhibition induces reprogramming of the tumour immune microenvironment that promotes ICI in NASH-HCC.

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 it might be synthesized into more elegant ways of targeting these powerful and abundant effector immune cells in the clinic.5 58, 61-63, 67, 68), providing opportunities for therapeutic intervention. Indeed, CXCR2 inhibitors are being trialed in cancer patients (NCT04477343, NCT03161431, NCT03177187, PRIMUS003).Although the molecules regulating neutrophil expansion and recruitment to tumors are shared across the entire population, neutrophils can exhibit striking functional differences, and information on their diversity continues to emerge. The mechanisms by which neutrophils are polarized towards pro-or antitumor states primarily occurs through cytokines, such as TGFb, IFNb, IFNg, G-CSF and GM-CSF (10-13, 26, 50, 69). Tumor hypoxia is another important regulator of neutrophil phenotype and polarization, since counteracting hypoxia in an autochthonous mouse model of PTEN-driven uterine cancer decreases neutrophil-mediated cancer progression (70). The importance of neutrophil polarization and diversity in cancer has been recently reviewed elsewhere (1-3, 71, 72). However, it is important to mention that specific nomenclature describing neutrophil polarization states have led to confusion when comparing data in the field. These terms include N1/N2 neutrophils, which were coined to mirror T helper cell (Th)-1/2 immunity and M1/M2 macrophages; granulocytic or polymorphonuclear myeloid-derived suppressor cells (G/PMN-MDSCs), which are T cell-inhibiting neutrophils; as well as low-density neutrophils (LDNs) and high-density neutrophils (HDNs), whose name stems from the location of neutrophils in density gradients. There are many biological arguments for and against the continued use of these terms (1, 2, 73, 74), but overall, we argue that to more accurately describe emerging data in the field they should be avoided. The terms above are either too narrow or too simplistic in their ability to capture the inherent plasticity of neutrophils, or they perpetuate the incorrect notion that N1, N2, MDSCs, LDNs, HDNs are cell populations distinct from neutrophils. These terms describe pathological activation or maturation states of neutrophils, rather than separate cell types (75). Neutrophil participation in metastasisT...

Highlights d Eosinophils release extracellular DNA traps and inhibit microfilariae motility d Microfilariae-induced eosinophil extracellular DNA traps are a conserved mechanism d Microfilariae trigger DNA release by eosinophils through the dectin-1 receptor d Microfilariae coated with DNA traps are cleared more efficiently in vivo

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.

EGR2 controls alveolar macrophage function in health and disease.

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.

PLOS NEGLECTED TROPICAL DISEASESPLOS Neglected Tropical Diseases | https://doi.