Philip L. Simonian

γδ T cells protect against lung fibrosis via IL-22

Inflammation-induced pulmonary fibrosis (PF) leads to irreversible loss of lung function and is a predictor of mortality in numerous lung diseases. Why some subjects with lung inflammation but not others develop PF is unclear. In a mouse model of hypersensitivity pneumonitis that progresses to lung fibrosis upon repeated exposure to the ubiquitous microorganism Bacillus subtilis, γδ T cells expand in the lung and inhibit collagen deposition. We show that a subset of these γδ cells represents the predominant source of the Th17 cytokine IL-22 in this model. Preventing expression of IL-22, either by mutating the aryl hydrocarbon receptor (AhR) or inhibiting AhR signaling, accelerated lung fibrosis. Direct blockade of IL-22 also enhanced collagen deposition in the lung, whereas administration of recombinant IL-22 inhibited lung fibrosis. Moreover, the presence of protective γδ T cells and IL-22 diminished recruitment of CD4+ T cells to lung. These data reveal a protective pathway that involves the inhibition of αβ T cells by regulatory IL-22–secreting γδ T cells.

IL-17A-Expressing T Cells Are Essential for Bacterial Clearance in a Murine Model of Hypersensitivity Pneumonitis

Hypersensitivity pneumonitis (HP) is an inflammatory lung disease characterized by a diffuse mononuclear cell infiltrate in the lung that can progress to pulmonary fibrosis with chronic exposure to an inhaled Ag. We previously reported that C57BL/6 mice repeatedly exposed to the ubiquitous microorganism Bacillus subtilis develop mononuclear infiltrates in the lung that contain Vγ6/Vδ1+ γδ T cells. In the absence of this T cell subset, mice treated with B. subtilis had significantly increased collagen deposition in the lung, suggesting a regulatory role for Vγ6/Vδ1+ γδ T cells. To further investigate the role of Vγ6/Vδ1+ γδ T cells in B. subtilis-induced lung fibrosis, we exposed transgenic Vγ6/Vδ1 mice to this microorganism and found decreased collagen content in the lung compared with wild-type C57BL/6 mice. Cytokine analysis of lung homogenates from wild-type C57BL/6 mice demonstrated increased IL-17A concentrations with repeated exposure to B. subtilis. In the absence of IL-17 receptor signaling, IL-17ra−/− mice had delayed clearance of B. subtilis with increased lung inflammation and fibrosis. Although IL-17A was predominantly expressed by Vγ6/Vδ1+ T cells, a compensatory increase in IL-17A expression by CD4+ T cells was seen in the absence of γδ T cells that resulted in similar levels of IL-17A in the lungs of TCRδ−/− and wild-type C57BL/6 mice. In combination, our data suggest an important role for IL-17A-expressing T lymphocytes, both γδ and αβ T cells, in eliminating this microorganism that prevents excessive inflammation and eventual lung fibrosis in this murine model of B. subtilis-induced hypersensitivity pneumonitis.

Bak can accelerate chemotherapy-induced cell death independently of its heterodimerization with Bcl-XL and Bcl-2.

Bak has been shown to both promote apoptosis and to inhibit cell death while two other members of the Bcl-2 family of proteins, Bcl-XL and Bcl-2 delay apoptosis induced by various stimuli including chemotherapeutic agents. We generated clones with stable expression of Bak wild-type (wt) and Bak with its BH3 (Δ78-86) domain deleted (ΔBH3) in FL5.12 cells or FL5.12 cells expressing either Bcl-XL or Bcl-2 to determine if Bak could accelerate apoptosis and antagonize the death repressor activity of Bcl-XL and Bcl-2 during chemotherapy-induced apoptosis. We found that Bak accelerated cell death in FL5.12 cells treated with etoposide, fluorouracil or taxol. In FL5.12 cells expressing Bcl-XL and Bak wt or Bak ΔBH3, both Bak wt or Bak ΔBH3 were able to antagonize the protective effect of Bcl-XL when treated with etoposide or fluorouracil. Bak wt or Bak ΔBH3 were also able to abrogate the protective effect of Bcl-2 in cells expressing Bcl-2 and Bak wt or Bak ΔBH3 when challenged by etoposide or fluorouracil. Immunoprecipitation studies revealed that deletion of BH3 disrupted heterodimerization between Bak and Bcl-XL and that both Bak wt and Bak ΔBH3 failed to interact with Bcl-2. These results demonstrate that Bak does not require its BH3 domain to promote apoptosis in stably transfected cells. Furthermore, Bak can accelerate chemotherapy-induced cell death independently of its heterodimerization with Bcl-XL and Bcl-2.

Bax Homodimerization Is Not Required for Bax to Accelerate Chemotherapy-induced Cell Death

Bax, a member of the Bcl-2 family of proteins, has been shown to accelerate apoptosis induced by growth factor withdrawal, γ-irradiation, and the chemotherapeutic agent, etoposide. The mechanism by which Bax promotes apoptosis is poorly understood. Bax forms homodimers which have been suggested to act as accelerators or inducers of cell death. However, the requirement for homodimerization of Bax to promote cell death remains unclear. We performed site-directed mutagenesis of the BH1, BH2, and BH3 in Bax to determine the regions of Bax required for homodimerization and to define the role of Bax homodimers in cell death induced by chemotherapy drugs. Bax proteins expressing alanine substitutions of the highly conserved amino acids glycine 108 (G108) in BH1, tryptophan 158 (W158) in BH2, and glycine 67 and aspartic acid 68 (GD67-68) in BH3 as well as deletion of the most conserved amino acids in BH1 (Δ102-112) and BH2 (Δ151-159) and deletion of BH3 (Δ63-71) maintained their ability to accelerate chemotherapy-induced cell death. Immunoprecipitation studies revealed that Bax with deletions in BH1 and BH2 still associated with wild-type Bax while deletion of BH3 disrupted Bax homodimerization. These results demonstrate that Bax does not require the conserved regions of homology, BH1, BH2, or BH3, to accelerate chemotherapy-induced cell death. Furthermore, our results established BH3 as a region required for Bax homodimerization in mammalian cells and demonstrate that monomeric forms of Bax are active in accelerating cell death induced by chemotherapy agents.

Regulatory T cells modulate granulomatous inflammation in an HLA-DP2 transgenic murine model of beryllium-induced disease

Significance Genetic linkage to major histocompatibility complex class II proteins has been observed in many immunological disorders; yet, little is known about the underlying mechanisms of these associations. For chronic beryllium disease (CBD), the linkage to HLA-DPB1 alleles encoding a glutamic acid at position 69 of the β-chain is well established. We tested whether the presence of HLA-DP2 is sufficient for the development of a disease-specific model of CBD. HLA-DP2 transgenic mice developed a beryllium-specific adaptive immune response composed of CD4+ T cells that secrete Th1-type cytokines and are HLA-DP2-restricted, thus replicating the major features of the human disease. In addition, regulatory T cells modulate granuloma formation in the lungs of beryllium oxide-exposed HLA-DP2 transgenic mice. Susceptibility to chronic beryllium disease (CBD) is linked to certain HLA-DP molecules, including HLA-DP2. To elucidate the molecular basis of this association, we exposed mice transgenic (Tg) for HLA-DP2 to beryllium oxide (BeO) via oropharyngeal aspiration. As opposed to WT mice, BeO-exposed HLA-DP2 Tg mice developed mononuclear infiltrates in a peribronchovascular distribution that were composed of CD4+ T cells and included regulatory T (Treg) cells. Beryllium-responsive, HLA-DP2–restricted CD4+ T cells expressing IFN-γ and IL-2 were present in BeO-exposed HLA-DP2 Tg mice and not in WT mice. Using Be-loaded HLA-DP2–peptide tetramers, we identified Be-specific CD4+ T cells in the mouse lung that recognize identical ligands as CD4+ T cells derived from the human lung. Importantly, a subset of HLA-DP2 tetramer-binding CD4+ T cells expressed forkhead box P3, consistent with the expansion of antigen-specific Treg cells. Depletion of Treg cells in BeO-exposed HLA-DP2 Tg mice exacerbated lung inflammation and enhanced granuloma formation. These findings document, for the first time to our knowledge, the development of a Be-specific adaptive immune response in mice expressing HLA-DP2 and the ability of Treg cells to modulate the beryllium-induced granulomatous immune response.

REGULATION OF LYMPHOID APOPTOSIS BY Bcl-2 AND Bcl-xL

Naturally occurring cell death is common during lymphoid development and activation. The death of developing lymphoid cells is a highly regulated process that serves to select lymphoid populations that are functionally competent, and to remove cells that are not longer needed or potentially autoreactivel. Elimination of self-reactive B and T lymphocytes by apoptosis is thought to play a major role in the establishment of self-tolerance. The latter process is mediated by high avidity interactions between antigen receptors and self-antigens2. In contrast, signaling via the Fas receptor appears to play a major role in the elimination of activated lymphocytes during immune responses in peripheral tissues3. In addition, to the antigen and Fas receptors, survival of lymphocytes is controlled by certain cytokines and costimulatory signals4−5. The intracellular mechanism that regulates and executes the death program is still poorly understood but it is thought that cell death is controlled by a genetic program induced within the dying lymphocyte. Recently, several genes have been identified that appear to play critical roles in lymphoid survival6. The bc1-2 protooncogene was the first member of a growing family of genes that suppresses cell death in lymphoid cells7. Constitutive expression of bc1-2 in lymphoid cells prevents or delays apoptosis induced by multiple stimuli7. A role of Bc1-2 in T and B-cell biology was suggested by its highly restricted cellular distribution during development and in mature lymphoid populations8−9. Recent evidences suggest that Bc1-2 plays a role in positive selection of thymocytes 10−12. However, the ability of Bc1-2 to influence negative selection of thymocytes and immature B cells is controversial13−15.

IL-17-Producing γδ T Cells in Auto-immune Disease

In both mice and humans, γδ T cells often represent a significant source of IL-17. Here we review the evidence that IL-17-producing γδ T cells contribute to auto-immune disease and discuss the role that they play in disease processes, including their influence on the development or activity of TH17 αβ T cells. Although IL-17-producing γδ T cells clearly can exacerbate auto-immune disease, in some systems they have instead been shown to play a protective role. The ability to produce IL-22 as well may be critical for this protective role.

Role of γδ T Cells in Lung Inflammation.

The resident population of γδ T cells in the normal lung is small but during lung inflammation, γδ T cells can increase dramatically. Histological analysis reveals diverse interactions between γδ T cells and other pulmonary leukocytes. Studies in animal models show that γδ T cells play a role in allergic lung inflammation where they can protect normal lung function, that they also are capable of resolving infection-induced pulmonary inflammation, and that they can help preventing pulmonary fibrosis. Lung inflammation threatens vital lung functions. Protection of the lung tissues and their functions during inflammation is the net-effect of opposing influences of specialized subsets of γδ T cells as well as interactions of these cells with other pulmonary leukocytes.