ITK independent development of Th17 responses during hypersensitivity pneumonitis driven lung inflammation

Abstract

T helper 17 (Th17) cells develop in response to T cell receptor signals (TCR) in the presence of specific environments, and produce the inflammatory cytokine IL17A. These cells have been implicated in a number of inflammatory diseases and represent a potential target for ameliorating such diseases. The kinase ITK, a critical regulator of TCR signals, has been shown to be required for the development of Th17 cells. However, we show here that lung inflammation induced by Saccharopolyspora rectivirgula (SR) induced Hypersensitivity pneumonitis (SR-HP) results in a neutrophil independent, and ITK independent Th17 responses, although ITK signals are required for γδ T cell production of IL17A. Transcriptomic analysis of resultant ITK independent Th17 cells suggest that the SR-HP-induced extrinsic inflammatory signals may override intrinsic T cell signals downstream of ITK to rescue Th17 responses in the absence of ITK. These findings suggest that the ability to pharmaceutically target ITK to suppress Th17 responses may be dependent on the type of inflammation.

Fig. 1. ITK is not required for the development of SR-induced hypersensitivity pneumonitis.

a WT or Itk^−/− mice were exposed to PBS or SR intranasally 3×/week over 3 weeks and lung sections were analyzed by H&E staining. b Lungs from SR-exposed WT mice were analyzed for the indicated mRNA by qRT-PCR (filled squares: IFNγ; filled triangles: IL4 and filled circles: IL17A, n = 3, *p = 0.0025 vs. time 0, **p = 0.007 vs. time 0 for IL17A). c WT or Itk^−/− mice were exposed to SR as in (a) and lungs analyzed for total cell numbers of γδ T cells (filled circles: WT γδ T cells; open circles Itk^−/− γδ T cells, n = 6 for WT group; 3–6 for Itk^−/− group), d αβ T cells (filled circles: WT αβ T cells; open circles Itk^−/− αβ T cells, (n = 3/group), e CD4⁺ and CD8⁺ T cells (filled circles: WT CD4⁺ T cells; open circles Itk^−/− CD4⁺ T cells; filled triangles: WT CD8⁺ T cells; open triangles Itk^−/− CD8⁺ T cells, (n = 3–6/group), or f neutrophils (filled circles: WT; open circles Itk^−/−, n = 3–4/group).

Fig. 2. αβ T cells are the major producers of IL17A during the development of SR-induced hypersensitivity pneumonitis and ITK is not required for their ability to produce IL17A.

a WT non-IL17A-GFP reporter mice, or WT or Itk^−/− IL17A-GFP mice were exposed to SR as in Fig. 1, and frozen lung sections were analyzed for IL17A-GFP by fluorescence microscopy, Blue = DAPI staining, Green = IL17A-GFP⁺ cells indicated by white arrows. b WT IL17A-GFP mice were exposed to SR as in Fig. 1, and lung cells that are GFP⁺ were gated (i.e., GFP⁺ > cell type) and analyzed for numbers of αβ (filled circles), γδ (filled boxes) T cells and neutrophils (filled diamonds). (n = 3/group). c Proportion of GFP⁺ lung cells (i.e., GFP⁺ > cell type) from SR-exposed WT (filled symbols) or Itk^−/− (open symbols) IL17A-GFP mice that are αβ (circles), γδ (squares) T cells and neutrophils (diamonds) (n = 3/group). d WT (filled symbols) or Itk^−/− (open symbols) IL17A-GFP mice were exposed to SR as in Fig. 1, and αβ (circles) and γδ (squares) T cells were analyzed for the proportion that is IL17A-GFP⁺ (i.e., cell type > IL17A-GFP⁺) (n = 3–4/group). e Lung CD4⁺ (circles) and CD8⁺ αβ (triangles) T cells were analyzed for proportion IL17A-GFP⁺ (i.e., cell type > IL17A-GFP⁺) (n = 3–4/group).

Fig. 3. Neutrophils are not required for the recruitment of T cells or their production of IL17A during the development of SR-induced hypersensitivity pneumonitis.

WT IL17A-GFP mice were injected with anti-Ly6G (1A8) or rat IgG_2a prior to the first SR exposure, then subsequently every other day for 14 days in concert with SR exposure. Flow cytometric analysis of a neutrophils (left panel) and proportion and number of neutrophils determined (right panels) (n = 2 for IgG_2a group, open boxes; n = 3 for 1A8 group, filled triangles, *p = 0.005 for proportion and *p = 0.01 for number of neutrophils between IgG_2a and 1A8). b αβ and γδ T cells, c CD4⁺ T cells. d, e Flow cytometric analysis of IL17A-GFP expression in d αβ CD4⁺ cells, e γδ T cells (n = 2 for IgG_2a group, open boxes; n = 3 for 1A8 group, filled triangles).

Fig. 4. Itk^−/− T cells receive strong signals in vivo during the development of SR-induced hypersensitivity pneumonitis.

a CD4⁺ T cells from non-exposed WT (filled circles) or Itk^−/− (open circles) Nurr77-GFP mice were analyzed for expression of Nurr77-GFP and MFI plotted (n = 4 for WT group; n = 6 for Itk^−/− group, *p = 1.1e−7). b WT (filled circles) or Itk^−/− (open circles) Nurr77-GFP mice were exposed to SR as in Fig. 1, and lung CD4⁺ T cells were analyzed for expression of Nurr77-GFP and MFI plotted (n = 3/group, *p = 0.0006). c Expression of Nr4a1 (gene for Nurr77) determined from RNA-sequencing (FKPM) (data from cells used for RNA-sequencing analysis depicted in Fig. 5, WT (filled circles) or Itk^−/− (open circles), n = 3/group, *p = 4.8e−5).

Fig. 5. Transcriptomic analysis of SR-induced Th17 cells.

WT or Itk^−/− IL17A-GFP/Foxp3-RFP mice were exposed to SR as in Fig. 1 and lung IL17A-GFP⁺/Foxp3^-/CD4⁺ αβ T cells sort purified, and RNA sequenced. a PCA plot of the transcriptome of SR-induced WT (orange circles) or Itk^−/− (green circles) Th17 cells. Axes show the principal components with the greatest difference (PC1 vs. PC3). b Heat map and hierarchical clustering of the transcriptome of SR-induced WT or Itk^−/− Th17 cells. c Volcano plot of transcripts that are significantly different (>2-fold) between SR-induced WT or Itk^−/− Th17 cells. d Heat map of Th17 related transcription factor expression between SR-induced WT or Itk^−/− Th17 cells. e Heat map of Th17 associated cytokine expression between SR-induced WT or Itk^−/− Th17 cells GSEA plots of pathways that are significantly positively or negatively enriched between SR-induced WT or Itk^−/− Th17 cells. f GSEA plots of pathways that are significantly positively or negatively enriched between SR-induced WT or Itk^−/− Th17 cells. g PCA plot of the transcriptome of SR-induced WT(orange circles) or Itk^−/− (green circles) Th17 cells using custom signature Th17 gene set. Axes show the principal components with the greatest difference (PC1 vs. PC3). h Heat map and hierarchical clustering of the transcriptome of SR-induced WT or Itk^−/− Th17 cells using custom signature Th17 gene set. i) Volcano plot of using custom Th17 gene set transcripts that are significantly different (>2-fold) between SR-induced WT or Itk^−/− Th17 cells.

Fig. 6. ATAC-Seq analysis of SR-induced Th17 cells.

WT (orange tracks) or Itk^−/− (green tracks) IL17A-GFP mice were exposed to SR as in Fig. 1 and lung IL17A-GFP⁺/Foxp3-RFP^-/CD4⁺ αβ T cells sort purified, and processed as described for ATAC-Seq. a Tracks of Tnfr8 and Zbt32 loci from WT or Itk^−/− Th17 cells. b Tracks of the loci of IL6 and TGFβ related transcription factors Stat3, Smad2, and Smad3. c Tracks of the loci of Th17 related transcriptional factors Nfatc1, Junb, Runx1, Hif1a, Irf4, Batf, and Maf. d Tracks of the loci of Th17 related cytokines Il17a, Il17f, Il21, and Il22.