Healthy and Patient Type 2 Innate Lymphoid Cells are Differently Affected by in vitro Culture Conditions

Innate lymphoid cells (ILCs) are critical mediators of immunological and physiological responses at mucosal barrier sites. Whereas neurotransmitters can stimulate ILCs, the synthesis of small-molecule neurotransmitters by these cells has only recently been appreciated. Group 2 ILCs (ILC2s) are shown here to synthesize and release acetylcholine (ACh) during parasitic nematode infection. The cholinergic phenotype of pulmonary ILC2s was associated with their activation state, could be induced by in vivo exposure to extracts of Alternaria alternata or the alarmin cytokines interleukin-33 (IL-33) and IL-25, and was augmented by IL-2 in vitro. Genetic disruption of ACh synthesis by murine ILC2s resulted in increased parasite burdens, lower numbers of ILC2s, and reduced lung and gut barrier responses to Nippostrongylus brasiliensis infection. These data demonstrate a functional role for ILC2-derived ACh in the expansion of ILC2s for maximal induction of type 2 immunity.

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IL-9–mediated survival of type 2 innate lymphoid cells promotes damage control in helminth-induced lung inflammation

Journal of Experimental Medicine ◽

10.1084/jem.20130071 ◽

2013 ◽

Vol 210 (13) ◽

pp. 2951-2965 ◽

Cited By ~ 239

Author(s):

Jan-Eric Turner ◽

Peter J. Morrison ◽

Christoph Wilhelm ◽

Mark Wilson ◽

Helena Ahlfors ◽

...

Keyword(s):

Lung Inflammation ◽

Damage Control ◽

Recovery Phase ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Th2 Cells ◽

Nippostrongylus Brasiliensis

IL-9 fate reporter mice established type 2 innate lymphoid cells (ILC2s) as major producers of this cytokine in vivo. Here we focus on the role of IL-9 and ILC2s during the lung stage of infection with Nippostrongylus brasiliensis, which results in substantial tissue damage. IL-9 receptor (IL-9R)–deficient mice displayed reduced numbers of ILC2s in the lung after infection, resulting in impaired IL-5, IL-13, and amphiregulin levels, despite undiminished numbers of Th2 cells. As a consequence, the restoration of tissue integrity and lung function was strongly impaired in the absence of IL-9 signaling. ILC2s, in contrast to Th2 cells, expressed high levels of the IL-9R, and IL-9 signaling was crucial for the survival of activated ILC2s in vitro. Furthermore, ILC2s in the lungs of infected mice required the IL-9R to up-regulate the antiapoptotic protein BCL-3 in vivo. This highlights a unique role for IL-9 as an autocrine amplifier of ILC2 function, promoting tissue repair in the recovery phase after helminth-induced lung inflammation.

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The transcription factor Bcl11b is specifically expressed in group 2 innate lymphoid cells and is essential for their development

Journal of Experimental Medicine ◽

10.1084/jem.20142318 ◽

2015 ◽

Vol 212 (6) ◽

pp. 865-874 ◽

Cited By ~ 94

Author(s):

Yong Yu ◽

Cui Wang ◽

Simon Clare ◽

Juexuan Wang ◽

Song-Choon Lee ◽

...

Keyword(s):

Transcription Factor ◽

Cell Lineage ◽

Influenza Virus Infection ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Effector Cytokines ◽

Group 2

Group 2 innate lymphoid cells (ILCs), or ILC2s, are a subset of recently identified ILCs, which play important roles in innate immunity by producing type 2 effector cytokines. Several transcription factors have been found to have critical functions in the development of both ILC2s and T cells. We report here that Bcl11b, a transcription factor essential in T cell lineage commitment and maintenance, is specifically expressed in progenitors committed to the ILC2 lineage and is required for ILC2 development. The Bcl11b gene is expressed in ∼28% of ILC progenitors (ILCPs; common helper innate lymphoid progenitors or ILCPs expressing either ID2 or promyelocytic leukemia zinc finger, respectively). Both in vitro and in vivo, these Bcl11b-expressing early ILCPs generate only ILC2s. Inactivation of Bcl11b causes a complete loss of ILC2 development from hematopoietic progenitors, which is confirmed upon immune challenge with either papain administration or influenza virus infection.

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Serum free culture for the expansion and study of type 2 innate lymphoid cells

Scientific Reports ◽

10.1038/s41598-021-91500-z ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Pablo de Lucía Finkel ◽

Christopher Sherwood ◽

Iryna Saranchova ◽

Wenjing Xia ◽

Lonna Munro ◽

...

Keyword(s):

Current Knowledge ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Serum Free ◽

Successful Isolation ◽

Potential Applications ◽

Rare Cells ◽

First Time

AbstractType 2 innate lymphoid cells (ILC2s) were discovered approximately ten years ago and their clinical relevance is gaining greater importance. However, their successful isolation from mammalian tissues and in vitro culture and expansion continues to pose challenges. This is partly due to their scarcity compared to other leukocyte populations, but also because our current knowledge of ILC2 biology is incomplete. This study is focused on ST2+ IL-25Rlo lung resident ILC2s and demonstrate for the first time a methodology allowing mouse type 2 innate lymphoid cells to be cultured, and their numbers expanded in serum-free medium supplemented with Interleukins IL-33, IL-2, IL-7 and TSLP. The procedures described methods to isolate ILC2s and support their growth for up to a week while maintaining their phenotype. During this time, they significantly expand from low to high cell concentrations. Furthermore, for the first time, sub-cultures of primary ILC2 purifications in larger 24- and 6-well plates were undertaken in order to compare their growth in other media. In culture, ILC2s had doubling times of 21 h, a growth rate of 0.032 h−1 and could be sub-cultured in early or late phases of exponential growth. These studies form the basis for expanding ILC2 populations that will facilitate the study and potential applications of these rare cells under defined, serum-free conditions.

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Pulmonary type-2 innate lymphoid cells in paediatric severe asthma: phenotype and response to steroids

European Respiratory Journal ◽

10.1183/13993003.01809-2018 ◽

2019 ◽

Vol 54 (2) ◽

pp. 1801809 ◽

Cited By ~ 11

Author(s):

Prasad Nagakumar ◽

Franz Puttur ◽

Lisa G. Gregory ◽

Laura Denney ◽

Louise Fleming ◽

...

Keyword(s):

Symptom Control ◽

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Th2 Cells ◽

Poor Control ◽

Inhaled Steroids ◽

Elevated Type

Children with severe therapy-resistant asthma (STRA) have poor control despite maximal treatment, while those with difficult asthma (DA) have poor control from failure to implement basic management, including adherence to therapy. Although recognised as clinically distinct, the airway molecular phenotype, including the role of innate lymphoid cells (ILCs) and their response to steroids in DA and STRA is unknown.Immunophenotyping of sputum and blood ILCs and T-cells from STRA, DA and non-asthmatic controls was undertaken. Leukocytes were analysed longitudinally pre- and post-intramuscular triamcinolone in children with STRA. Cultured ILCs were evaluated to assess steroid responsiveness in vitro.Airway eosinophils, type 2 T-helper (Th2) cells and ILC2s were significantly higher in STRA patients compared to DA and disease controls, while IL-17+ lymphoid cells were similar. ILC2s and Th2 cells were significantly reduced in vivo following intramuscular triamcinolone and in vitro with steroids. Furthermore, asthma attacks and symptoms reduced after systemic steroids despite persistence of steroid-resistant IL-17+ cells and eosinophils.Paediatric STRA and DA have distinct airway molecular phenotypes with STRA characterised by elevated type-2 cells. Systemic corticosteroids, but not maintenance inhaled steroids resulted in improved symptom control and exacerbations concomitant with a reduction in functional ILC2s despite persistently elevated IL-17+ lymphoid cells.

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Roles of Type 1, 2, and 3 Innate Lymphoid Cells in Herpes Simplex Virus 1 InfectionIn VitroandIn Vivo

Journal of Virology ◽

10.1128/jvi.00523-19 ◽

2019 ◽

Vol 93 (13) ◽

Cited By ~ 3

Author(s):

Satoshi Hirose ◽

Shaohui Wang ◽

Kati Tormanen ◽

Yizhou Wang ◽

Jie Tang ◽

...

Keyword(s):

Innate Lymphoid Cells ◽

Lymphoid Cells ◽

Wild Type ◽

Herpes Simplex Virus 1 ◽

Type 3 ◽

Deficient Mice ◽

Hsv 1

ABSTRACTInnate lymphoid cells (ILCs) play important roles in host defense and inflammation. They are classified into three distinct groups based on their cytokine and chemokine secretion patterns and transcriptome profiles. Here, we show that ILCs isolated from mice can be infected with herpes simplex virus 1 (HSV-1) but that subsequent replication of the virus is compromised. After infection, type 2 ILCs expressed significantly higher levels of granulocyte colony-stimulating factor (G-CSF), interleukin 1α (IL-1α), IL-6, IL-9, RANTES, tumor necrosis factor alpha (TNF-α), CXCL1, CXCL2, CXCL10, CCL3, and CCL4 than infected type 1 or type 3 ILCs. Transcriptome-sequencing (RNA-seq) analysis of the ILCs 24 h after HSV-1 infection revealed that 77 herpesvirus genes were detected in the infected type 3 ILCs, whereas only 11 herpesvirus genes were detected in infected type 1 ILCs and 27 in infected type 2 ILCs. Compared with uninfected cells, significant upregulation of over 4,000 genes was seen in the HSV-1-infected type 3 ILCs, whereas 414 were upregulated in the infected type 1 ILCs and 128 in the infected type 2 ILCs. In contrast, in all three cell types, only a limited number of genes were significantly downregulated. Type 1, type 2, and type 3 ILC-deficient mice were used to gain insights into the effects of the ILCs on the outcome of ocular HSV-1 infection. No significant differences were found on comparison with similarly infected wild-type mice or on comparison of the three strains of deficient mice in terms of virus replication in the eyes, levels of corneal scarring, latency-reactivation in the trigeminal ganglia, or T-cell exhaustion. Although there were no significant differences in the survival rates of infected ILC-deficient mice and wild-type mice, there was significantly reduced survival of the infected type 1 or type 3 ILC-deficient mice compared with type 2 ILC-deficient mice. Adoptive transfer of wild-type T cells did not alter survival or any other parameters tested in the infected mice. Our results indicate that type 1, 2, and 3 ILCs respond differently to HSV-1 infectionin vitroand that the absence of type 1 or type 3, but not type 2, ILCs affects the survival of ocularly infected mice.IMPORTANCEIn this study, we investigated for the first time what roles, if any, innate lymphoid cells (ILCs) play in HSV-1 infection. Analysis of isolated ILCsin vitrorevealed that all three subtypes could be infected with HSV-1 but that they were resistant to replication. The expression profiles of HSV-1-induced cytokines/chemokines and cellular and viral genes differed among the infected type 1, 2, and 3 ILCsin vitro. While ILCs play no role or a redundant role in the outcomes of latency-reactivation in infected mice, absence of type 1 and type 3, but not type 2, ILCs affects the survival of infected mice.

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