Characterization of the MT-2 Treg-like cell line in the presence and absence of forkhead box P3 (FOXP3).

CD4+ forkhead box P3 (FOXP3)+ regulatory T cells (Tregs) are essential in maintaining immune tolerance and suppressing excessive immune responses. Tregs also contribute to tissue repair processes distinct from their roles in immune suppression. For these reasons, Tregs are candidates for targeted therapies for inflammatory and autoimmune diseases, and in diseases where tissue damage occurs. MT-2 cells, an immortalized Treg-like cell line, offer a model to study Treg biology and their therapeutic potential. In the present study, we use clustered regularly interspaced palindromic repeats (CRISPR)-mediated knockdown of FOXP3 in MT-2 cells to understand the transcriptional and functional changes that occur when FOXP3 is lost and to compare MT-2 cells with primary human Tregs. We demonstrate that loss of FOXP3 affects the transcriptome of MT-2 cells and that FOXP3's potential downstream targets include a wide range of transcripts that participate in the cell cycle, promote growth and contribute to inflammatory processes, but do not wholly simulate previously reported human primary Treg transcriptional changes in the absence of FOXP3. We also demonstrate that FOXP3 regulates cell cycling and proliferation, expression of molecules crucial to Treg function and MT-2 cell-suppressive activities. Thus, MT-2 cells offer opportunities to address regulatory T-cell functions in vitro.

Comparison of different methods of initiating lung inflammation and the sex-specific effects on inflammatory parameters.

Sex as a biological variable is an essential element of preclinical research. Sex-specific differences in lung volume, alveolar number, body weight, and the relationship between lung and body weight result in important questions about generating equivalent injuries in males and females so that comparisons in their responses can be assessed. Few studies compare stimulus dosing methods for murine lung models investigating immune responses. To examine sex-specific effects, we explored several dosing techniques for three stimuli, LPS, Streptococcus pneumoniae, and influenza A, on survival, injury parameters in bronchoalveolar lavage (BAL), and immune cell numbers in single-cell lung suspensions after injury. These data demonstrate that body weight-based dosing produced fewer differences between sexes when compared with injury initiated with inocula containing the same number of organisms. Comparison of the lung and body weights showed that females had a greater lung-to-body weight ratio than males. However, in LPS-induced injury, adjusting the dose for sex differences in this ratio in addition to body weight provided no new information about sex differences compared with dosing by body weight alone, most likely due to the variability in measures of the immune response. Studies evaluating BAL volumes revealed that smaller but more lavages resulted in greater returns and lower protein concentrations, particularly in the smaller female lungs. Thus, designing dosing and measurement methods that generate equivalent injuries facilitates comparison of immune responses between sexes. Continued development of methods for both induction and evaluation of injury will likely facilitate identification of sex differences in immune responses.

Bronchoalveolar Tregs are associated with duration of mechanical ventilation in acute respiratory distress syndrome.

BACKGROUND: Foxp3+ regulatory T cells (Tregs) play essential roles in immune homeostasis and repair of damaged lung tissue. We hypothesized that patients whose lung injury resolves quickly, as measured by time to liberation from mechanical ventilation, have a higher percentage of Tregs amongst CD4+ T cells in either airway, bronchoalveolar lavage (BAL) or peripheral blood samples. METHODS: We prospectively enrolled patients with ARDS requiring mechanical ventilation and collected serial samples, the first within 72 h of ARDS diagnosis (day 0) and the second 48-96 h later (day 3). We analyzed immune cell populations and cytokines in BAL, tracheal aspirates and peripheral blood, as well as cytokines in plasma, obtained at the time of bronchoscopy. The study cohort was divided into fast resolvers (FR; n = 8) and slow resolvers (SR; n = 5), based on the median number of days until first extubation for all participants (n = 13). The primary measure was the percentage of CD4+ T cells that were Tregs. RESULTS: The BAL of FR contained more Tregs than SR. This finding did not extend to Tregs in tracheal aspirates or blood. BAL Tregs expressed more of the full-length FOXP3 than a splice variant missing exon 2 compared to Tregs in simultaneously obtained peripheral blood. CONCLUSION: Tregs are present in the bronchoalveolar space during ARDS. A greater percentage of CD4+ cells were Tregs in the BAL of FR than SR. Tregs may play a role in the resolution of ARDS, and enhancing their numbers or functions may be a therapeutic target.

Baseline and innate immune response characterization of a Zfp30 knockout mouse strain.

Airway neutrophilia is correlated with disease severity in a number of chronic and acute pulmonary diseases, and dysregulation of neutrophil chemotaxis can lead to host tissue damage. The gene Zfp30 was previously identified as a candidate regulator of neutrophil recruitment to the lungs and secretion of CXCL1, a potent neutrophil chemokine, in a genome-wide mapping study using the Collaborative Cross. ZFP30 is a putative transcriptional repressor with a KRAB domain capable of inducing heterochromatin formation. Using a CRISPR-mediated knockout mouse model, we investigated the role that Zfp30 plays in recruitment of neutrophils to the lung using models of allergic airway disease and acute lung injury. We found that the Zfp30 null allele did not affect CXCL1 secretion or neutrophil recruitment to the lungs in response to various innate immune stimuli. Intriguingly, despite the lack of neutrophil phenotype, we found there was a significant reduction in the proportion of live Zfp30 homozygous female mutant mice produced from heterozygous matings. This deviation from the expected Mendelian ratios implicates Zfp30 in fertility or embryonic development. Overall, our results indicate that Zfp30 is an essential gene but does not influence neutrophilic inflammation in this particular knockout model.

Impact of Regulatory T Cells on Type 2 Alveolar Epithelial Cell Transcriptomes during Resolution of Acute Lung Injury and Contributions of IFN-γ.

By enhancing tissue repair and modulating immune responses, Foxp3+ regulatory T cells (Tregs) play essential roles in resolution from lung injury. The current study investigated the effects that Tregs exert directly or indirectly on the transcriptional profiles of type 2 alveolar epithelial (AT2) cells during resolution in an experimental model of acute lung injury. Purified AT2 cells were isolated from uninjured mice or mice recovering from LPS-induced lung injury, either in the presence of Tregs or in Treg-depleted mice, and transcriptome profiling identified differentially expressed genes. Depletion of Tregs resulted in altered expression of 49 genes within AT2 cells during resolution, suggesting that Tregs present in this microenvironment influence AT2-cell function. Biological processes from Gene Ontology enriched in the absence of Tregs included those describing responses to IFN. Neutralizing IFN-γ in Treg-depleted mice reversed the effect of Treg depletion on inflammatory macrophages and B cells by preventing the increase in inflammatory macrophages and the decrease in B cells. Our results provide insight into the effects of Tregs on AT2 cells. Tregs directly or indirectly impact many AT2-cell functions, including IFN type I and II-mediated signaling pathways. Inhibition of IFN-γ expression and/or function may be one mechanism through which Tregs accelerate resolution after acute lung injury.

Effects of IFN-γ on immune cell kinetics during the resolution of acute lung injury.

The immunologic responses that occur early in the acute respiratory distress syndrome (ARDS) elicit immune-mediated damage. The mechanisms underlying the resolution of ARDS, particularly the role of signaling molecules in regulating immune cell kinetics, remain important questions. Th1-mediated responses can contribute to the pathogenesis of acute lung injury (ALI). Interferon-gamma (IFN-γ) orchestrates early inflammatory events, enhancing immune-mediated damage. The current study investigated IFN-γ during resolution in several experimental models of ALI. The absence of IFN-γ resulted in altered kinetics of lymphocyte and macrophage responses, suggesting that IFN-γ present in this microenvironment is influential in ALI resolution. Genetic deficiency of IFN-γ or administering neutralizing IFN-γ antibodies accelerated the pace of resolution. Neutralizing IFN-γ decreased the numbers of interstitial and inflammatory macrophages and increased alveolar macrophage numbers during resolution. Our results underline the complexity of lung injury resolution and provide insight into the effects through which altered IFN-γ concentrations affect immune cell kinetics and the rate of resolution. These findings suggest that therapies that spatially or temporally control IFN-γ signaling may promote ALI resolution. Identifying and elucidating the mechanisms critical to ALI resolution will allow the development of therapeutic approaches to minimize collateral tissue damage without adversely altering the response to injury.

Transcriptional analysis of Foxp3+ Tregs and functions of two identified molecules during resolution of ALI.

Recovery from acute lung injury (ALI) is an active process. Foxp3+ Tregs contribute to recovery from ALI through modulating immune responses and enhancing alveolar epithelial proliferation and tissue repair. The current study investigates Treg transcriptional profiles during resolution of ALI in mice. Tregs from either lung or splenic tissue were isolated from uninjured mice or mice recovering from ALI and then examined for differential gene expression between these conditions. In mice with ALI, Tregs isolated from the lungs had hundreds of differentially expressed transcripts compared with those from the spleen, indicating that organ specificity and microenvironment are critical in Treg function. These regulated transcripts suggest which intracellular signaling pathways modulate Treg behavior. Interestingly, several transcripts having no prior recognized function in Tregs were differentially expressed by lung Tregs during resolution. Further investigation into 2 identified transcripts, Mmp12 and Sik1, revealed that Treg-specific expression of each plays a role in Treg-promoted ALI resolution. This study provides potentially novel information describing the signals that may expand resident Tregs, recruit or retain them to the lung during ALI, and modulate their function. The results provide insight into both tissue- and immune microenvironment-specific transcriptional differences through which Tregs direct their effects.

Foxp3+ Regulatory T Cell Expression of Keratinocyte Growth Factor Enhances Lung Epithelial Proliferation.

Repair of the lung epithelium after injury is a critical component for resolution; however, the processes necessary to drive epithelial resolution are not clearly defined. Published data demonstrate that Foxp3+ regulatory T cells (Tregs) enhance alveolar epithelial proliferation after injury, and Tregs in vitro directly promote type II alveolar epithelial cell (AT2) proliferation, in part by a contact-independent mechanism. Therefore, we sought to determine the contribution of Treg-specific expression of a growth factor that is known to be important in lung repair, keratinocyte growth factor (kgf). The data demonstrate that Tregs express kgf and that Treg-specific expression of kgf regulates alveolar epithelial proliferation during the resolution phase of acute lung injury and in a model of regenerative alveologenesis in vivo. In vitro experiments demonstrate that AT2 cells cocultured with Tregs lacking kgf have decreased rates of proliferation compared with AT2 cells cocultured with wild-type Tregs. Moreover, Tregs isolated from lung tissue and grown in culture express higher levels of two growth factors that are important for lung repair (kgf and amphiregulin) compared with Tregs isolated from splenic tissue. Lastly, Tregs isolated from human lung tissue can be stimulated ex vivo to induce kgf expression. This study reveals mechanisms by which Tregs direct tissue-reparative effects during resolution after acute lung injury, further supporting the emerging role of Tregs in tissue repair.