Interleukin-2-Inducible T-Cell Kinase Deficiency Impairs Early Pulmonary Protection Against Mycobacterium tuberculosis Infection

Interleukin-2 (IL-2) inducible T-cell kinase (ITK) is a non-receptor tyrosine kinase highly expressed in T-cell lineages and regulates multiple aspects of T-cell development and function, mainly through its function downstream of the T-cell receptor. Itk deficiency can lead to CD4 lymphopenia and Epstein-Bar virus (EBV)-associated lymphoproliferation and recurrent pulmonary infections in humans. However, the role of the ITK signaling pathway in pulmonary responses in active tuberculosis due to Mtb infection is not known. We show here that human lungs with active tuberculosis exhibit altered T-cell receptor/ITK signaling and that Itk deficiency impaired early protection against Mtb in mice, accompanied by defective development of IL-17A-producing γδ T cells in the lungs. These findings have important implications of human genetics associated with susceptibility to Mtb due to altered immune responses and molecular signals modulating host immunity that controls Mtb activity. Enhancing ITK signaling pathways may be an alternative strategy to target Mtb infection, especially in cases with highly virulent strains in which IL-17A plays an essential protective role.


INTRODUCTION
Mycobacterium tuberculosis (Mtb) is the causative agent of what remains one of the most insidious and invasive human infections, responsible for millions of cases of active lung diseases and deaths per year in the world [WHO Global Tuberculosis Report, (1) and O'Garra et al. (2)]. The immune responses by macrophages, neutrophils, and T-cell populations contribute to protective immunity controlling Mtb expansion in the host and transmission to others (2,3). Genetic and environmental factors of the host associated with primary and acquired immunodeficiency can lead to an increased risk of developing active tuberculosis that presents severe pulmonary illness in the clinic (2,4). Our knowledge of the molecular pathways of innate and adaptive immune effector functions remains incomplete, and a better understanding of potential host factors underlying the pulmonary complications could lead to the development of more innovative therapeutic strategies.
Epidemiological analysis of single-nucleotide polymorphism has also revealed a connection between greater ITK promoter activity and higher risk of asthmatic incidence in humans, which might be associated with the function of ITK in promoting T-cell activation (50). In murine models of allergic asthma, the expression of ITK is critical for the activation and development of Th2 and Th17 cells and the associated airway and tracheal inflammation (40,51). Interestingly, a genome-wide association study of susceptibility to Mycobacterium avium subspecies paratuberculosis in Holstein cattle identified chromosomal regions that included the ITK gene (52). However, the role of ITK signaling pathway in pulmonary responses in active tuberculosis due to Mtb infection is unknown.
Here, we show that the TCR/ITK signaling pathway is enriched in human lungs with active tuberculosis and that Itk deficiency impaired early protection against Mtb in mice, accompanied by defective development of IL-17A-producing γδ T cells in the lungs. Furthermore, ITK appears to regulate the dynamics of lung myeloid cells, which may further contribute to immune control of Mtb at the early stage of infection.

Mice
All mice were on the C57BL/6 background. Both female and male mice at the age of 6-12 weeks were used. All experiments were approved by the Office of Research Protections Institutional Animal Care and Use Committee at Cornell University.

Microarray and Data Analyses
Microarray data from lung normal tissue and caseous granulomas from active tuberculosis (TB) patient was generated as previously described (53,54). Microarray data is available in the Gene Expression Omnibus under accession number GSE20050. In brief, tissues were fixed, and areas of interest were dissected using laser capture microdissection on the Leica AS LMD system (Leica, Buffalo Grove, IL). Total RNA was isolated and used on the GeneChip Human X3P Array (Affymetrix, Santa Clare, CA) following the manufacturer's instruction. Data analysis was performed in R (version 3.5.3) and Bioconductor (version 3.8). Probe intensities were log 2 transformed and median centered. Differentially expressed genes were identified with limma (version 3.38.3) (55). In the case of multiple probes mapped to a gene, the probe with the maximum fold change was selected to represent the gene. Gene set enrichment analyses (GSEA) to determine over-and underrepresented gene sets were performed using the Kyoto Encyclopedia of Genes and Genomes pathway database as reference in gage (version 2.32.1) (56). All pathways that exhibited an up-or downregulated trend in caseum samples compared to normal tissues are summarized in the Supplementary Material (Supplementary Table 1). Enrichment score and core genes that drive the score are determined using the GSEA platform developed by the Broad Institute (57). Visualization of TCR signaling pathway with differential gene expression was performed in pathview (version 1.

Mtb Infection and Colony-Forming Unit Counts
Mice were inoculated intranasally with ∼1,000 CFUs of Erdman Mtb constitutively expressing mCherry (mCherry-Mtb) (59) in 25 µl of phosphate-buffered saline (PBS) containing 0.05% Tween-80. Mice were euthanized after 2 and 4 weeks of infection. The left lung lobe and the accessory lobe of the right lung were removed and homogenized in PBS containing 0.05% Tween-80. Bacterial loads were determined by plating serial dilutions of the homogenates on 7H10 agar plates.

Histology and Pathogenic Scoring
Lung samples were fixed in 4% paraformaldehyde overnight, followed by hematoxylin and eosin (H&E) staining. Histological images were analyzed using DP2-BSW software (Olympus, Waltham, MA) to quantify the percentage of affected tissue area and score the severity of pathology (60).

Isolation of Lung Cells
Mice were euthanized at the indicated time points, and lungs were aseptically removed. To obtain a singlecell suspension, lungs were minced and digested in 5% fetal bovine serum/PBS solution containing 250 U/ml collagenase IV (Worthington, Lakewood, NJ) and 20 U/ml DNase (Roche, Indianapolis, IN) for 30 min at 37 • C. Lung digestions were then passed through a 70µm cell strainer, and red blood cells were lysed with ammonium-chloride-potassium buffer.

Flow Cytometry
Surface protein staining was done with antibodies for surface markers in PBS, in the presence of Fc Block (BioLegend) and fixable viability dye (Tonbo Biosciences). To determine cytokine production, cells were stimulated as indicated, followed by surface staining, then were fixed with 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA), and permeabilized and stained with antibodies in PBS containing 0.3% saponin FIGURE 1 | ITK expression is altered in human lungs with active TB. (A) Pathway enrichment analyses of human lung tissues with active TB ("Caseum," N = 5), compared to normal lung tissues ("Normal," N = 2). Pathway enrichment statistical values were for pathways enriched in "Caseum" group were shown as positive values (upregulated in "Caseum" compared to "Normal"), while those enriched in normal lungs were shown as negative values (upregulated in "Normal" compared to "Caseum"). Twenty most enriched pathways in the "Caseum" group (out of 145 pathways with FDR < 0.05), and 10 most enriched pathways in "Normal" group (out of 11 pathways with FDR < 0.05) are shown. Red arrow indicates T-cell receptor signaling pathway. (B) Gene set enrichment score, profile of T-cell signaling receptor pathway and heatmap of the levels of expression of the score-driving genes in T-cell receptor signaling pathway. Red arrow indicates ITK. FDR, false discover rate.
(Sigma). To stain for nuclear transcription factors Foxp3 and Ki67, following surface staining, cells were fixed, permeabilized, and stained using Foxp3 staining buffer set (eBioscience). All flow cytometry data were acquired on LSRII (BD Biosciences) and analyzed in FlowJo (Tree Star, Ashland, OR).

Statistical Analysis
Two-tailed Student's t-test and two-way analysis of variance (ANOVA) between groups were performed using Prism (GraphPad, San Diego, CA), with p < 0.05 considered statistically significant. "NS" indicates differences that are not significant.

TCR/ITK Signaling Components Are Upregulated in Active Tuberculosis in Humans
The progression of human active TB disease and transmission involves the development of the caseous granuloma, in which both Mtb and the immune response are active (53). We have previously isolated human granulomata from patients with active TB (Caseum) and analyzed the transcriptomic profile in comparison to uninvolved lung tissue (normal) (53). Using pathway enrichment analyses, we found that genes of the TCR signaling were significantly enriched in the caseum tissue that was subjected to active TB, compared to uninvolved lung tissue (Figure 1). Among the enrichment score-driving critical genes of the TCR signaling, the levels of transcripts for ITK and its signaling components (5) such as LCK, GRB2, SLP76, NCK1, FYN, and PLCG are significantly upregulated in caseated granulomas compared to uninvolved lung tissue ( Figure 1B). Among the genes that are significantly enriched in active TB, ITK locates in the hub of the TCR signaling pathway (Supplementary Figure 1). These data imply a role for ITK signaling in host immune activity during active TB.

Clearance and Increased Lung Pathology
Given the observation that the TCR signaling pathway was upregulated in the face of active TB in human lungs and that ITK is a critical score-driving gene for the pathway enrichment (Figure 1), we sought to determine the role of ITK in host responses to Mtb infection. In murine models of Itk deficiency, despite no difference in animal mortality, we found that Mtb bacterial burden was significantly higher in the lungs in the absence of ITK 4 weeks post-Mtb infection (Figure 2A). Moreover, pulmonary pathology was elevated in the absence of ITK, with significantly larger areas in the airway affected at higher pathological scores ( Figure 2B). Notably, compared to wild-type (WT) mice, the relative kinetics of bacterial growth in the lungs of Itk −/− and Rag −/− mice are similar (61,62). These data suggest that the TCR/ITK signaling pathway regulates immune responses that contribute to limiting Mtb growth and controlling pulmonary inflammation 4 weeks postinfection.

Itk-Deficient Mice Exhibit Altered Early Immune Responses to Mtb Infection
The progression of human active TB disease and transmission involves the development of both innate and adaptive immunity. The relative abundance of lung phagocyte populations is extremely dynamic at the early stage of Mtb infection (63). Importantly, the various phagocytes in the lung provide different environments for Mtb and reveal distinct permissiveness for the growth of Mtb (64,65). We thus analyzed populations of innate immune cells in both WT and Itk-deficient mice infected with Mtb that constitutively express fluorescent protein mCherry, which allows mapping of the cellular location of intracellular bacteria. During early-stage infection, we  observed increased total numbers of neutrophils and alveolar macrophages in the lung at 2 weeks postinfection in both WT and Itk −/− mice ( Figure 3A). Further analysis of the pulmonary immune cell populations revealed that, in the absence of ITK, the proportion of Mtb-infected alveolar and interstitial macrophages was significantly higher early after infection (2 weeks); in addition, the proportion of Mtbinfected neutrophils was significantly higher 4 weeks after infection (Figure 3). Effective removal of Mtb-infected apoptotic neutrophils by macrophages, or efferocytosis, is considered beneficial for host defense (66). The failed clearance of infected neutrophils in Itk-deficient mice at 4 weeks suggests that Itk may be involved in regulating efferocytosis of lung macrophages. Both alveolar macrophages and neutrophils have been demonstrated as permissive cell types in Mtb infection by providing a hospitable environment for optimal bacterial growth (64,65). The increased proportion of Mtbinfected neutrophils in the absence of ITK is associated with the increased bacterial burden at 4 weeks, suggesting that bacterial burden may be attributed to this difference. Therefore, these data suggest that ITK regulates the dynamic of lung phagocytes and contributes to host protection against Mtb infection.

ITK is highly expressed in T-cell lineages including γδ and αβ T cells. To further determine whether the absence of ITK affects T-cell and other related lymphocyte responses during
Mtb infection, we also analyzed the abundance of γδ T cells, NK cells, NKT cells, and αβ T cells, including total αβ T cells, CD4 + conventional αβ T cells, CD8 + αβ T cells, and CD4 + Foxp3 + regulatory T (Treg) cells. Comparable numbers of γδ T cells, NKT cells, and CD8 + αβ T cells were observed in WT and Itk-deficient mice infected with Mtb, while NK cells and CD4 + αβ T cells were significantly increased, and Foxp3 + Treg cells were significantly reduced in the absence of ITK (Figure 4). These results seemed surprising, as a reduced number of Treg cells accompanied by increased numbers of CD4 + αβ T cells may suggest a more active immune response. The hosts' ability to limit bacterial growth and control pulmonary inflammation was, however, impaired in the absence of ITK. A possible explanation could be that the effector activity of the lymphocytes observed in the infected airway against Mtb may differ. These effector immune responses could involve innate lymphocyte activities, as well as Mtb antigen-specific adaptive lymphocyte functions.

ITK Is Critical for γδ T-Cell-Derived IL-17A Production During Mtb Infection
It has been reported that IL-17A is protective during primary infection of virulent Mtb (67), and IL-17A is predominantly produced by γδ T cells in the lungs early after Mtb infection (68). Indeed, while we detected significant IL-17A production by γδ T cells in the lungs of WT mice infected with Mtb (red foreground in Figures 5A-C), very limited IL-17A was produced by the conventional CD4 + αβ Th17 cells (gray background in Figure 5A, as well as in Figure 5D). Interestingly, in the absence of ITK, there was a marked reduction in IL-17A-producing γδ T cells in the lung (Figures 5A-C). In contrast to the significant levels of IL-17A production in γδ T cells, IL-17A production by CD4 + and CD8 + αβ T cells during Mtb infection was minimal (Figure 5B vs. Figure 5D). Furthermore, in the absence of ITK, unlike γδ T-cell-derived IL-17A that was significantly impaired, IL-17A production by CD4 + and CD8 + αβ T cells did not exhibit overt differences, although the proportion of those cells making IL-17A was quite low (Figure 5D).

Itk Deficiency Has Minimal Impact on Antigen-Specific αβ T-Cell Responses During Mtb Infection
Our data above supports a strong role of ITK in promoting the IL-17A-producing effector γδ T cells during Mtb infection. It is possible that αβ T-cell effector functions were also altered in the absence of ITK and further contributed to the impaired bacterial clearance and enhanced lung pathology as observed in Figure 2. To determine whether ITK regulates αβ T-cell effector function, we stimulated cells isolated from the lungs of the infected mice 4 weeks postinfection. We found that bulk T-cell activation by PMA and ionomycin suggested that ITK is not required for CD4 + and CD8 + αβ T cells to produce effector cytokines tumor necrosis factor alpha (TNF-α) and interferongamma (IFN-γ) during Mtb infection ( Figure 6A). Moreover, to our surprise, stimulation of Mtb antigen-specific CD4 + αβ T cells with ESAT-6 4−17 (MHCII-restricted epitope), or CD8 + αβ T cells with TB10.4 4−11 (MHCI-restricted epitope) revealed no difference in production of TNF-α and IFN-γ by these cells in the absence of ITK (Figure 6B). Along with the data above, our results suggest that the major protective role of ITK during Mtb infection might be executed through ITK-mediated IL-17A production by γδ T cells.

Itk Regulates γδ T Cell but Not αβ T-Cell Proliferation During Mtb Infection
Itk deficiency led to significantly impaired effector γδ T cells (Figure 5) but not αβ T cells (Figure 6). These might be the results of differential requirement of ITK signaling in Tcell expansion. Using proliferative maker Ki67 to detect T-cell proliferation, we found that Mtb-driven early proliferation of γδ T cells was severely impaired in ITK deficiency (Figure 7, top panel), but not the CD4 + and CD8 + αβ T-cell subsets (Figure 7, middle and bottom panels). These data, in part, explain the selective defect of effector γδ T-cell development in Itk-deficient mice during Mtb infection.

DISCUSSION
The immune cells that control Mtb include macrophages, neutrophils, and T-cell populations (2,3). The TCR pathway is elevated in active Mtb infection in human lung, and ITK is a major component of this pathway; however, its role in the T-cell response to Mtb is not known. Here, we demonstrate a protective role of ITK in Mtb infections in murine models, with a particular role in the ability of lung γδ T cells to produce IL-17A, which is associated with Mtb residence in lung neutrophils.
These findings have important implications for understanding the T-cell immune response to Mtb and the role of ITK in this process.
In humans, Mtb infects via inhalation of a low dose of aerosolized bacteria; therefore, low-dose aerosol inoculation would better mimic the nature of Mtb infection in humans. However, in the absence of the ability to perform aerosolized infections, the standard experimental protocol of Mtb infection in mice is 1,000 CFU via intranasal inoculation (69)(70)(71)(72). Monocytes play an essential role in initiating T-cell responses in the lung against Mtb infection (73), and using this standard protocol of Mtb infection in mice, we previously observed monocytosis (64), similar to what has been reported in other studies using aerosolized low-dose infection protocols (74,75). Using intranasal inoculation with 1,000 CFU of Mtb in mice, we observed that the numbers of host myeloid cells infiltrating to the lung during Mtb infection are significantly altered in the absence of ITK. Alveolar macrophages are considered as the preferred replicating niche for Mtb and promote the early  stage of infection (64,76). In Itk-deficient mice, there are more alveolar macrophages harboring Mtb, suggesting a more permissive cellular environment in the lung. Moreover, although fewer neutrophils are present in the lung in the absence of ITK, there are more Mtb resident in neutrophils at 4 weeks postinfection. The redistribution of Mtb in different phagocytes in the absence of ITK appears to correlate with the increased bacterial burden in the lung 4 weeks postinfection. Other than T cells, ITK has been indicated to regulate functions of innate immune cells such as mast cells (77,78). However, potential intrinsic functions of ITK in lung macrophages and neutrophils during Mtb infection would merit further studies.
Activation of γδ T cells for IL-17A production is severely impaired in Itk-deficient mice and very likely responsible for the resultant neutrophil recruitment early after infection(s), as well as the increased neutrophil resident Mtb population later in infection. Indeed, we have shown that ITK can regulate γδ T cell development and function in mouse (48), and while there was no apparent difference in peripheral blood γδ T-cell numbers in humans carrying ITK mutations (12), the normal range for γδ T cells varies quite widely by anatomical location, as well as by geography and ethnicity (79). Despite relatively normal antigenspecific αβ T-cell responses, our results identify ITK signaling as an essential player for the IL-17A production by γδ T cells, the predominant source of IL-17A in Mtb-infected lungs (68). ITK exhibited a γδ T cell-specific function, as compared to αβ T cells, in driving T-cell proliferation during early immune responses to Mtb infections. Our work suggests that ITK deficiency in humans may lead to γδ T-cell deficiency in expansion and production of IL-17A, in the face of Mtb infection.
We and others have previously reported a role for ITK signaling in regulating Foxp3 + regulatory T (Treg) cell development and function (41,80). In the absence of ITK, the proportion of Treg cells among CD4 + T cells in the lymphoid organs of naive mice is increased, and the differentiation of inducible Treg cells from CD4 + naive T cells in vitro is also enhanced (41,80). The role of ITK in Treg cell responses in the lung during Mtb infection was, however, unclear. In mouse model of Mtb infection, we observed higher Mtb burdens, higher lung pathological scores and reduced numbers of Treg cells found in the lungs in Itk-deficient mice, compared to those in the infected WT mice (Figure 4). Tissue damage may be explained by excessive growth of the pathogen and/or immunopathology. Indeed, we observed impaired γδ T cell expansion and γδ T cellderived IL-17A production in Mtb-infected Itk-deficient mice but no overt differences in other immune effectors. The decrease in Treg numbers in the lung during Mtb infection in the absence of ITK may explain higher levels of immunopathology potentially due to impaired immunomodulatory function. Future research using murine models with conditional deletion of Itk specifically in Foxp3 + Treg cells would allow more in-depth investigation of the role of ITK in Treg cells during Mtb infection.
Taken together, these findings suggest a potential role for ITK in active TB in humans, in addition to its known connection with primary immunodeficiency, susceptibility to EBV, lymphoproliferative diseases, and asthma (12,50,51). These findings also have important implications for human genetics associated with susceptibility to Mtb due to altered immune responses and molecular signals modulating host immunity that controls the progression of active tuberculosis.
Our findings support a role of ITK signaling in promoting protective immune responses against Mtb, in particular, γδ T cell expansion and production of IL-17A, which could contribute to the modulation of tuberculosis, especially in infections with highly virulent bacterial strains in which IL-17A has been shown to play an essential protective role (67). However, our work also sounds a note of caution for the potential use of compounds such as Ibrutinib that inhibit the related kinase BTK as well as ITK (81). Given the potential for inhibition of ITK, patients being treated with Ibrutinib may need to be monitored for infection or potential reactivation of latent Mtb.

DATA AVAILABILITY STATEMENT
The datasets generated for this study can be found in the Gene Expression Omnibus under accession number GSE20050.

ETHICS STATEMENT
The animal study was reviewed and approved by the Institutional Animal Care and Use Committees at Cornell University.

AUTHOR CONTRIBUTIONS
LH, MM, NN, JE, CL, and WH performed the experiments. LH, TS, AA, and WH analyzed and interpreted data. LH, AA, and WH wrote the manuscript. KY performed bioinformatic analyses. DR contributed reagents and intellectual input. AA and WH conceived research and designed experiments.

FUNDING
This work was supported in part by grants from the National Institutes of Health (AI120701 and AI138570 to AA, AI129422 to AA and WH, GM130555 Sub-6610 to WH, and AI134183 to DR), and a Pilot Grant (to WH) from LSU-Tulane COBRE Center for Experimental Infectious Disease Research (funded by NIH P30GM110760). JE was supported by a training program in Biomedical and Bioengineering at Cornell University (funded by NIH T32EB023860). MM is a recipient of the Pathobiological Sciences Graduate Program Fellowship in the School of Veterinary Medicine at the Louisiana State University.

ACKNOWLEDGMENTS
We thank Amie Redko for the animal care and Ling Zhang for the technical assistance.