Resistance to apoptosis underpins the corticosteroid insensitivity of group 2 innate lymphoid cells

Abstract

TSLP.To test the hypothesis that absence of keratins in KtyI 2/2 cells upregulates TSLP through stimulation of EGFR, growth factor-starved WT and KtyI 2/2 cells were treated for 3 and 7 hours with 200 ng/mL recombinant AREG, followed by TSLP mRNA and protein analysis. This revealed a strong induction of Tslp mRNA in WT and KtyI 2/2 cells caused by the AREG stimulus (Fig 2 , A), with greater Tslp mRNA levels in KtyI 2/2 cells. Interestingly, AREG-EGFR stimulation of TSLP protein by 4-fold was restricted to KtyI 2/2 cells, indicating increased sensitization of keratin-deficient cells toward TSLP induction through EGFR signaling (Fig 2 , B). Furthermore, we show that stable re-expression of keratin 14 using lentiviral transduction in keratin-free keratinocytes prevents a strong induction of TSLP by AREG compared with green fluorescent protein (GFP)-expressing keratinocytes, suggesting that keratins repress an AREG-EGFR-TSLP signaling pathway (Fig 2 , C).To substantiate the finding that AREG-mediated TSLP induction depended on EGFR and ERK1/2 (Fig 2, A and B), Western blot analysis of Raf and ERK1/2 kinases was performed. This confirmed their involvement in AREG-mediated TSLP induction (Fig 2 , D). Nuclear factor kB signaling, as measured based on p65 phosphorylation, remained unaltered between WT and KtyI 2/2 cells and was not induced upon AREG-EGFR stimulation (Fig 2 , D9). This suggests that nuclear factor kB is not significantly involved in the keratin-mediated TSLP induction in keratinocytes. Intriguingly, mRNA levels of the EGFR-regulated cytokine CCL20, which are known to be increased in blister fluid from patients with EBS, 7 were also upregulated strongly after AREG-EGFR stimulation in KtyI 2/2 keratinocytes (Fig 2, E). Other inflammatory mediators, such as CCL2, CXCL1, CXCL10, IL-1A, Defensin b4, and S100A7 remained uninduced or were regulated equally on AREG-EGFR stimulation in WT and KtyI 2/2 cells (see Fig E1 , B, in this article's Online Repository at www.jacionline.org). This indicates that lack of keratins triggers expression of a distinct set of cytokines, chemokines, and antimicrobial peptides at the level of AREG-EGFR to induce a distinct inflammatory response.To examine whether AREG is also involved in TSLP upregulation in patients with EBS, AREG and TSLP serum protein levels in sera from patients with EBS were analyzed (Fig 2 , F). Six of 7 tested patients with EBS had increased TSLP serum protein levels (> _50 pg/mL), of whom 3 (50%) exhibited detectable AREG serum protein levels. Moreover, increased epidermal AREG levels were recently identified in a microarray analysis of 6 patients with EBS, 8 which is in line with the known release of AREG from wound-activated keratinocytes. 9 We hypothesize that increased AREG levels in patients with EBS can trigger TSLP upregulation in keratinocytes with keratin defects to drive itch and T H 2 inflammation in patients with EBS.Collectively, we present a new mechanism for TSLP induction in keratin-deficient keratinocytes mediated by an autocrine AREG-EGFR...

Group 3 innate lymphoid cells secret neutrophil chemoattractants and are insensitive to glucocorticoid via aberrant GR phosphorylation

“…Patients with neutrophilic asthma usually have poor response to corticosteroids [ 34 – 36 ], and our previous study has found that the numbers of ILC3s in asthma patients were not reduced after treatment with prednisolone [ 23 ]. Therefore, we speculated that ILC3s might contribute to the steroid resistance.…”

“…The cells were cultured in RPMI with 10% human serum, 1× l -glutamine, 1× penicillin/streptomycin, 1× sodium pyruvate, 1× nonessential amino acids, and 250 U/mL IL-2. Details were described in our previously published study [ 23 ].…”

Inhibition of multipotent ILC2s by JAK3 inhibitor attenuates steroid-resistant asthma

“…Some limited evidence supports the notion that ILC2s may help mediate steroid-resistant asthma. Thus, Nagakumar et al found that steroid-resistant asthma patients bear more sputum ILC2s than control asthma patients 9 , while Luo et al reported circulating ILC2s from steroid-resistant asthma patients rose after steroid treatment, unlike Th2 cells 34 . Moreover, van der Ploeg et al observed that asthma patients have more in ammatory CD45RO + ILC2s in the in amed mucosal tissues and blood of asthma patients than controls and that these ILC2 frequencies correlate with disease severity and steroid resistance.…”

“…Luo et al found that peripheral blood ILC2s from healthy subjects express higher levels of the antiapoptotic genes BCL2 and BCL2L1 and lower levels of the pro-apoptotic genes BAX1 and BAK1 than Th2 cells. Since the ILC2s but not the Th2 cells were resistant to steroids, this suggests that ILC2s are less sensitive to the pro-apoptotic effects of steroids than Th2 cells 34 and TSLP together convert CD45RA + ILC2s, which are steroid-sensitive, into steroid-resistant CD45RO + ILC2s in vitro; TSLP on its own did not have this effect 35 . Kabata et al observed that steroid-sensitive murine ILC2s became steroid-resistant when they were stimulated in vitro with both IL-33 and TSLP 10 .…”

Chronic Allergen Challenge Induces Corticosteroid Insensitivity With Persistent Airway Remodeling and Type 2 Inflammation

“…IFNγ, or Th2 cell resistance to apoptosis ( Huang et al, 2001 ; Williams et al, 2018 ). Additionally, ILC2 activation, which promotes type 2 inflammation, could also contribute to enhanced allergic airway inflammation and corticosteroid insensitivity ( Luo et al, 2019 ). ILC2 regulation involves negative regulatory signals from IFNγ or PGE 2 ( Duerr et al, 2016 ; Maric et al, 2018 ; Zhou et al, 2018 ; Zhou et al, 2020 ).…”