Innate Lymphoid Cells are the Predominant Source of Interleukin-17A During the Early Pathogenesis of Acute Respiratory Distress Syndrome

Rationale: IL-17A is purported to help drive early pathogenesis in acute respiratory distress syndrome (ARDS) by enhancing neutrophil recruitment. Whilst IL-17A is the archetypal cytokine of T helper (Th)17 cells, it is produced by a number of lymphocytes, the source during ARDS being unknown.

Objectives: To identify the cellular source and the role of IL17A in the early phase of lung injury

Methods: Lung injury was induced in WT (C57BL/6) and IL-17 KO mice with aerosolised LPS (100 µg) or Pseudomonas aeruginosa infection. Detailed phenotyping of the cells expressing RORγt, the transcriptional regulator of IL-17 production, in the mouse lung at 24 hours was carried out by flow cytometry.

Measurement and Main Results: A 100-fold reduction in neutrophil infiltration was observed in the lungs of the IL-17A KO compared to wild type (WT) mice. The majority of RORγt+ cells in the mouse lung were the recently identified type 3 innate lymphoid cells (ILC3). Detailed characterisation revealed these pulmonary ILC3s (pILC3s) to be discrete from those described in the gut. The critical role of these cells was verified by inducing injury in Rag2 KO mice which lack T cells but retain ILCs. No amelioration of pathology was observed in the Rag2 KO mice.

Conclusions: IL-17 is rapidly produced during lung injury and significantly contributes to early immunopathogenesis. This is orchestrated largely by a distinct population of pILC3 cells. Modulation of pILC3s’ activity may potentiate early control of the inflammatory dysregulation seen in ARDS, opening up new therapeutic targets.

P2X1 regulated IL-22 secretion by innate lymphoid cells is required for efficient liver regeneration.

Paracrine signalling mediated via cytokine secretion is essential for liver regeneration after hepatic resection, yet the mechanisms of cellular crosstalk between immune and parenchymal cells are still elusive. Interleukin-22 (IL-22) is released by immune cells and mediates strong hepatoprotective functions. However, it remains unclear if IL-22 is critical for the crosstalk between liver lymphocytes and parenchymal cells during liver regeneration after partial hepatectomy. Here we found that plasma levels of IL-22 and its upstream cytokine IL-23 are highly elevated in patients after major liver resection. In a mouse model of partial hepatectomy, deletion of IL-22 was associated with significantly delayed hepatocellular proliferation and an increase of hepatocellular injury and endoplasmic reticulum stress. Using Rag1-/- and Rag2-/- γc-/- mice we show that the main producers of IL-22 post partial hepatectomy are conventional natural killer cells and innate lymphoid cells type 1. Extracellular ATP, a potent danger molecule, is elevated in patients immediately after major liver resection. Antagonism of the P2 type nucleotide receptors P2X1 and P2Y6 significantly decreased IL-22 secretion ex vivo. In vivo, specific inhibition of P2X1 was associated with decreased IL-22 secretion, elevated liver injury and impaired liver regeneration.

Basophils promote innate lymphoid cell responses in inflamed skin.

Type 2 inflammation underlies allergic diseases such as atopic dermatitis, which is characterized by the accumulation of basophils and group 2 innate lymphoid cells (ILC2s) in inflamed skin lesions. Although murine studies have demonstrated that cutaneous basophil and ILC2 responses are dependent on thymic stromal lymphopoietin, whether these cell populations interact to regulate the development of cutaneous type 2 inflammation is poorly defined. In this study, we identify that basophils and ILC2s significantly accumulate in inflamed human and murine skin and form clusters not observed in control skin. We demonstrate that murine basophil responses precede ILC2 responses and that basophils are the dominant IL-4-enhanced GFP-expressing cell type in inflamed skin. Furthermore, basophils and IL-4 were necessary for the optimal accumulation of ILC2s and induction of atopic dermatitis-like disease. We show that ILC2s express IL-4Rα and proliferate in an IL-4-dependent manner. Additionally, basophil-derived IL-4 was required for cutaneous ILC2 responses in vivo and directly regulated ILC2 proliferation ex vivo. Collectively, these data reveal a previously unrecognized role for basophil-derived IL-4 in promoting ILC2 responses during cutaneous inflammation.

Epithelial-intrinsic IKKα expression regulates group 3 innate lymphoid cell responses and antibacterial immunity

Innate lymphoid cells (ILCs) are critical for maintaining epithelial barrier integrity at mucosal surfaces; however, the tissue-specific factors that regulate ILC responses remain poorly characterized. Using mice with intestinal epithelial cell (IEC)-specific deletions in either inhibitor of κB kinase (IKK)α or IKKβ, two critical regulators of NFκB activation, we demonstrate that IEC-intrinsic IKKα expression selectively regulates group 3 ILC (ILC3)-dependent antibacterial immunity in the intestine. Although IKKβ(ΔIEC) mice efficiently controlled Citrobacter rodentium infection, IKKα(ΔIEC) mice exhibited severe intestinal inflammation, increased bacterial dissemination to peripheral organs, and increased host mortality. Consistent with weakened innate immunity to C. rodentium, IKKα(ΔIEC) mice displayed impaired IL-22 production by RORγt(+) ILC3s, and therapeutic delivery of rIL-22 or transfer of sort-purified IL-22-competent ILCs from control mice could protect IKKα(ΔIEC) mice from C. rodentium-induced morbidity. Defective ILC3 responses in IKKα(ΔIEC) mice were associated with overproduction of thymic stromal lymphopoietin (TSLP) by IECs, which negatively regulated IL-22 production by ILC3s and impaired innate immunity to C. rodentium. IEC-intrinsic IKKα expression was similarly critical for regulation of intestinal inflammation after chemically induced intestinal damage and colitis. Collectively, these data identify a previously unrecognized role for epithelial cell-intrinsic IKKα expression and TSLP in regulating ILC3 responses required to maintain intestinal barrier immunity.

Influence of Mid-Trimester Amniotic Fluid on Endogenous and Lipopolysaccharide-Mediated Responses of Mononuclear Lymphoid Cells

Problem We evaluated the influence of amniotic fluid (AF) on immune mediator production by mononuclear leukocytes. Method of study Thirty mid-gestation AFs were incubated with peripheral blood mononuclear cells (PBMCs) in the presence or absence of lipopolysaccharide (LPS). Supernatants were tested for interleukin (IL) -6, 10, 12, 23, tumor necrosis factor-alpha (TNF-alpha) and monocyte chemotactic protein (MCP)-1. Results Endogenous mediator production was minimal or non-detectable. AF stimulated endogenous MCP-1, IL-6 and TNF-alpha release. In the presence of LPS, production of MCP-1 and IL-10 by PBMCs was enhanced eightto ninefold by AF. Release of IL-6 and IL-23 was enhanced less than twofold by the addition of AF while TNF-alpha production was unchanged. AF-stimulated mediator production was similar irrespective of pregnancy outcome. Conclusion Selective AF stimulation of LPS-mediated MCP-1 and IL-10 release may be a mechanism to promote antibody production and the influx of phagocytic cells to engulf pathogens while downregulating the production of pro-inflammatory cytokines.

Immunomodulatory effects exerted by Lactobacillus salivarius strains on innate immune cells

Despite increased application of commensal bacteria for attempting to improve the symptoms of a variety of inflammatory conditions, including inflammatory bowel diseases, diarrhoea and irritable bowel syndrome, therapeutic approaches that involve live bacteria are hampered by a limited understanding of bacterium-host interactions. Lactobacilli are natural inhabitants of the mammalian gastrointestinal tract and many lactobacilli are regarded as probiotics meaning that they exert a beneficial influence on the health status of their consumers. Modulation of immune responses is a plausible mechanism underlying these beneficial effects. The aim of this thesis was to investigate the effect of 33 Lactobacillus salivarius strains on the production of inflammatory cytokines from a variety of human and mouse immune cells. Induction of immune responses in vitro was shown to be bacterial- and mouse strain-dependent, cell type-dependent, blood donor-dependent and bacterial cell number-dependent. Collectively, these data suggest the importance of a case-by-case selection of candidate strains for their potential therapeutic application. Toll-like receptors (TLRs) recognize microbe-associated molecular patterns (MAMPs) and play a critical role in shaping microbial-specific innate and adaptive immune responses. Following ligand engagement, TLRs trigger a complex network of signalling that culminate in the production of inflammatory mediators. The investigation of the molecular mechanisms underlying the Lb. salivarius-host interaction resulted in the identification of a novel role for TLR2 in negatively regulating TLR4 signalling originated from subcellular compartments within macrophages. Notably, sustained activation of JAK/STAT cascade and M1-signature genes in TLR2-/- macrophages was ablated by selective TLR4 and JAK inhibitors and by absence of TLR4 in TLR2/4-/- cells. In addition, other negative regulators of TLR signalling triggered by Lb. salivarius strains were found to be the adapter molecules TIRAP and TRIF. Understanding negative regulation of TLR signalling may pave the way for the development of novel therapeutics to limit inflammation in multiple diseases.

Multifactorial interaction of growth factors on Penaeus monodon lymphoid cells and the impact of IGFs in DNA synthesis and metabolic activity in vitro

Development of continuous cell lines from shrimp is essential to investigate viral pathogens. Unfortunately, there is no valid cell line developed from crustaceans in general and shrimps in particular to address this issue. Lack of information on the requirements of cells in vitro limits the success of developing a cell line, where the microenvironment of a cell culture, provided by the growthmedium, is of prime importance. Screening and optimization of growth medium components based on statistical experimental designs have been widely used for improving the efficacy of cell culture media. Accordingly, we applied Plackett–Burman design and response surface methodology to study multifactorial interactions between the growth factors in shrimp cell culture medium and to identify the most important ones for growth of lymphoid cell culture from Penaeus monodon. The statistical screening and optimization indicated that insulin like growth factor-I (IGF-I) and insulin like growth factor-II (IGF-II) at concentrations of 100 and 150 ng ml-1, respectively, could significantly influence the metabolic activity and DNA synthesis of the lymphoid cells. An increase of 53 % metabolic activity and 24.8 % DNA synthesis could be obtained, which suggested that IGF-I and IGFII had critical roles in metabolic activity and DNA synthesis of shrimp lymphoid cells

A pro-inflammatory role of lymphoid cells in acute pleurisy in rats

The effect of viable splenic lymphoid cells and their constituents (filtrate) on carrageenan-induced acute pleurisy was investigated in rats. Suspensions of lymphoid cells administered intravenously to recipients just prior to initiation of pleurisy enhance both the volume of exudate and cell accumulation in the pleural cavity 3 h after the irritation. Similar results were observed when filtrate of disrupted lymphoid cells was injected either 30 or 5 min before the carrageenan, but not when administered 30 min afterwards. Suspensions of bone marrow cells, on the contrary, were ineffective in producing an enhancement of the parameters studied. When administered into the pleural cavity together with carrageenan, the lymphoid cell filtrate augmented the inflammatory response to the irritant. Nevertheless, it was ineffective, per se, to elicit any local change. It is suggested that lymphoid cells may play a pro-inflammatory role in the initiation of the process by enhancing both the fluid and the cellular components of inflammation.

Rapid downregulation of innate immune cells, interleukin-12 and interleukin-23 in generalized pustular psoriasis with infliximab in combination with acitretin

Generalized pustular psoriasis (GPP) is a severe inflammatory disease characterized by recurrent eruptions of sterile pustules on erythematous skin. Although tumor necrosis factor (TNF) antagonists may lead to a rapid resolution of GPP, the mechanism of action of these agents remains to be investigated. Here, we sought to evaluate markers of immune response in the skin of a patient who experienced a rapid amelioration of GPP after treatment with infliximab and acitretin.

Interferon-γ Induces Expression of MHC Class II on Intestinal Epithelial Cells and Protects Mice from Colitis

Immune responses against intestinal microbiota contribute to the pathogenesis of inflammatory bowel diseases (IBD) and involve CD4(+) T cells, which are activated by major histocompatibility complex class II (MHCII) molecules on antigen-presenting cells (APCs). However, it is largely unexplored how inflammation-induced MHCII expression by intestinal epithelial cells (IEC) affects CD4(+) T cell-mediated immunity or tolerance induction in vivo. Here, we investigated how epithelial MHCII expression is induced and how a deficiency in inducible epithelial MHCII expression alters susceptibility to colitis and the outcome of colon-specific immune responses. Colitis was induced in mice that lacked inducible expression of MHCII molecules on all nonhematopoietic cells, or specifically on IECs, by continuous infection with Helicobacter hepaticus and administration of interleukin (IL)-10 receptor-blocking antibodies (anti-IL10R mAb). To assess the role of interferon (IFN)-γ in inducing epithelial MHCII expression, the T cell adoptive transfer model of colitis was used. Abrogation of MHCII expression by nonhematopoietic cells or IECs induces colitis associated with increased colonic frequencies of innate immune cells and expression of proinflammatory cytokines. CD4(+) T-helper type (Th)1 cells - but not group 3 innate lymphoid cells (ILCs) or Th17 cells - are elevated, resulting in an unfavourably altered ratio between CD4(+) T cells and forkhead box P3 (FoxP3)(+) regulatory T (Treg) cells. IFN-γ produced mainly by CD4(+) T cells is required to upregulate MHCII expression by IECs. These results suggest that, in addition to its proinflammatory roles, IFN-γ exerts a critical anti-inflammatory function in the intestine which protects against colitis by inducing MHCII expression on IECs. This may explain the failure of anti-IFN-γ treatment to induce remission in IBD patients, despite the association of elevated IFN-γ and IBD.

Innate immune cells express IL-17A/F in acute generalized exanthematous pustulosis and generalized pustular psoriasis.

Acute generalized exanthematous pustulosis (AGEP) and generalized pustular psoriasis (GPP) are rare pustular skin disorders with systemic involvement. IL-17A/F is a proinflammatory cytokine involved in various neutrophilic inflammatory disorders. Here we show that IL-17A/F is highly expressed by innate immune cells such as neutrophils and mast cells in both AGEP and GPP.