Cutting Edge: Dual Function of PPARγ in CD11c+ Cells Ensures Immune Tolerance in the Airways.

The respiratory tract maintains immune homeostasis despite constant provocation by environmental Ags. Failure to induce tolerogenic responses to allergens incites allergic inflammation. Despite the understanding that APCs have a crucial role in maintaining immune tolerance, the underlying mechanisms are poorly understood. Using mice with a conditional deletion of peroxisome proliferator-activated receptor γ (PPARγ) in CD11c(+) cells, we show that PPARγ performs two critical functions in CD11c(+) cells to induce tolerance, thereby preserving immune homeostasis. First, PPARγ was crucial for the induction of retinaldehyde dehydrogenase (aldh1a2) selectively in CD103(+) dendritic cells, which we recently showed promotes Foxp3 expression in naive CD4(+) T cells. Second, in all CD11c(+) cells, PPARγ was required to suppress expression of the Th17-skewing cytokines IL-6 and IL-23p19. Also, lack of PPARγ in CD11c(+) cells induced p38 MAPK activity, which was recently linked to Th17 development. Thus, PPARγ favors immune tolerance by promoting regulatory T cell generation and blocking Th17 differentiation.

Cutting Edge: MMP-9 inhibits IL-23p19 expression in dendritic cells by targeting membrane stem cell factor affecting lung IL-17 response.

We reported previously that c-kit ligation by membrane-bound stem cell factor (mSCF) boosts IL-6 production in dendritic cells (DCs) and a Th17-immune response. However, Th17 establishment also requires heterodimeric IL-23, but the mechanisms that regulate IL-23 gene expression in DCs are not fully understood. We show that IL-23p19 gene expression in lung DCs is dependent on mSCF, which is regulated by the metalloproteinase MMP-9. Th1-inducing conditions enhanced MMP-9 activity, causing cleavage of mSCF, whereas the opposite was true for Th17-promoting conditions. In MMP-9(-/-) mice, a Th1-inducing condition could maintain mSCF and enhance IL-23p19 in DCs, promoting IL-17-producing CD4(+) T cells in the lung. Conversely, mSCF cleavage from bone marrow DCs in vitro by rMMP-9 led to reduced IL-23p19 expression under Th17-inducing conditions, with dampening of intracellular AKT phosphorylation. Collectively, these results show that the c-kit/mSCF/MMP-9 axis regulates IL-23 gene expression in DCs to control IL-17 production in the lung.

An adhesion protein of Salmonella enterica serovar Typhi is required for pathogenesis and potential target for vaccine development.

More than half of all Salmonella enterica serovar Typhi genes still remain unannotated. Although pathogenesis of S. Typhi is incompletely understood, treatment of typhoid fever is complicated by the emergence of drug resistance. Effectiveness of the currently available vaccines is also limited. In search of novel virulence proteins, we have identified several putative adhesins of S. Typhi through computational approaches. Our experiment shows that a 27-kDa outer membrane protein (T2544) plays a major role in bacterial adhesion to the host through high-affinity binding to laminin. Its role in bacterial pathogenesis is underscored by reduced systemic invasion and a 10-fold higher LD(50) of the mutant bacteria in mice. T2544 is strongly immunogenic as revealed by the detection of sustained high titers of serum IgG and intestinal secretory IgA in the immunized mice. In vitro, T2544 antiserum enhanced uptake and clearance of Salmonella by macrophages and augmented complement-mediated lysis, indicating a contribution of T2544-specific antibodies to the killing process. This correlates well with the observed protection of mice immunized with recombinant T2544 or passively immunized with T2544 antiserum against subsequent bacterial challenge, suggesting that T2544-specific antibodies are involved in protection. The present study describes an adhesion protein of S. Typhi that contributes to bacterial pathogenesis. Protective antibodies in mice, rapid seroconversion of naturally infected individuals with increasing titers of anti-T2544 IgG from acute to convalescent sera suggesting antibody response in humans, and wide distribution and conservation of the cell-surface adhesin in the clinical isolates of different Salmonella serovars make T2544 a potential vaccine candidate.

Immunomodulatory role of mitochondrial DAMPs: a missing link in pathology?

In accordance with the endosymbiotic theory, mitochondrial components bear characteristic prokaryotic signatures, which act as immunomodulatory molecules when released into the extramitochondrial compartment. These endogenous immune triggers, called mitochondrial damage-associated molecular patterns (mtDAMPs), have been implicated in the pathogenesis of various diseases, yet their role remains largely unexplored. In this review, we summarise the available literature on mtDAMPs in diseases, with a special focus on respiratory diseases. We highlight the need to bolster mtDAMP research using a multipronged approach, to study their effect on specific cell types, receptors and machinery in pathologies. We emphasise the lacunae in the current understanding of mtDAMPs, particularly in their cellular release and the chemical modifications they undergo. Finally, we conclude by proposing additional effects of mtDAMPs in diseases, specifically their role in modulating the immune system.

The timing of differentiation and potency of CD8 effector function is set by RNA binding proteins.

CD8+ T cell differentiation into effector cells is initiated early after antigen encounter by signals from the T cell antigen receptor and costimulatory molecules. The molecular mechanisms that establish the timing and rate of differentiation however are not defined. Here we show that the RNA binding proteins (RBP) ZFP36 and ZFP36L1 limit the rate of differentiation of activated naïve CD8+ T cells and the potency of the resulting cytotoxic lymphocytes. The RBP function in an early and short temporal window to enforce dependency on costimulation via CD28 for full T cell activation and effector differentiation by directly binding mRNA of NF-κB, Irf8 and Notch1 transcription factors and cytokines, including Il2. Their absence in T cells, or the adoptive transfer of small numbers of CD8+ T cells lacking the RBP, promotes resilience to influenza A virus infection without immunopathology. These findings highlight ZFP36 and ZFP36L1 as nodes for the integration of the early T cell activation signals controlling the speed and quality of the CD8+ T cell response.

A genetically engineered, stem-cell-derived cellular vaccine.

Despite rapid clinical translation of COVID-19 vaccines in response to the global pandemic, an opportunity remains for vaccine technology innovation to address current limitations and meet challenges of inevitable future pandemics. We describe a universal vaccine cell (UVC) genetically engineered to mimic natural physiological immunity induced upon viral infection of host cells. Cells engineered to express the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike as a representative viral antigen induce robust neutralizing antibodies in immunized non-human primates. Similar titers generated in this established non-human primate (NHP) model have translated into protective human neutralizing antibody levels in SARS-CoV-2-vaccinated individuals. Animals vaccinated with ancestral spike antigens and subsequently challenged with SARS-CoV-2 Delta variant in a heterologous challenge have an approximately 3 log decrease in viral subgenomic RNA in the lungs. This cellular vaccine is designed as a scalable cell line with a modular poly-antigenic payload, allowing for rapid, large-scale clinical manufacturing and use in an evolving viral variant environment.

Multiomics analysis couples mRNA turnover and translational control of glutamine metabolism to the differentiation of the activated CD4+ T cell.

The ZFP36 family of RNA-binding proteins acts post-transcriptionally to repress translation and promote RNA decay. Studies of genes and pathways regulated by the ZFP36 family in CD4+ T cells have focussed largely on cytokines, but their impact on metabolic reprogramming and differentiation is unclear. Using CD4+ T cells lacking Zfp36 and Zfp36l1, we combined the quantification of mRNA transcription, stability, abundance and translation with crosslinking immunoprecipitation and metabolic profiling to determine how they regulate T cell metabolism and differentiation. Our results suggest that ZFP36 and ZFP36L1 act directly to limit the expression of genes driving anabolic processes by two distinct routes: by targeting transcription factors and by targeting transcripts encoding rate-limiting enzymes. These enzymes span numerous metabolic pathways including glycolysis, one-carbon metabolism and glutaminolysis. Direct binding and repression of transcripts encoding glutamine transporter SLC38A2 correlated with increased cellular glutamine content in ZFP36/ZFP36L1-deficient T cells. Increased conversion of glutamine to α-ketoglutarate in these cells was consistent with direct binding of ZFP36/ZFP36L1 to Gls (encoding glutaminase) and Glud1 (encoding glutamate dehydrogenase). We propose that ZFP36 and ZFP36L1 as well as glutamine and α-ketoglutarate are limiting factors for the acquisition of the cytotoxic CD4+ T cell fate. Our data implicate ZFP36 and ZFP36L1 in limiting glutamine anaplerosis and differentiation of activated CD4+ T cells, likely mediated by direct binding to transcripts of critical genes that drive these processes.

Cardiolipin-mediated PPARγ S112 phosphorylation impairs IL-10 production and inflammation resolution during bacterial pneumonia.

Bacterial pneumonia is a global healthcare burden, and unwarranted inflammation is suggested as an important cause of mortality. Optimum levels of the anti-inflammatory cytokine IL-10 are essential to reduce inflammation and improve survival in pneumonia. Elevated levels of the mitochondrial-DAMP cardiolipin (CL), reported in tracheal aspirates of pneumonia patients, have been shown to block IL-10 production from lung MDSCs. Although CL-mediated K107 SUMOylation of PPARγ has been suggested to impair this IL-10 production, the mechanism remains elusive. We identify PIAS2 to be the specific E3-SUMOligase responsible for this SUMOylation. Moreover, we identify a concomitant CL-mediated PPARγ S112 phosphorylation, mediated by JNK-MAPK, to be essential for PIAS2 recruitment. Furthermore, using a clinically tested peptide inhibitor targeting JNK-MAPK, we blocked these post-translational modifications (PTMs) of PPARγ and rescued IL-10 expression, improving survival in murine pneumonia models. Thus, we explore the mechanism of mito-DAMP-mediated impaired lung inflammation resolution and propose a therapeutic strategy targeting PPARγ PTMs.

Virus Induced Lymphocytes (VIL) as a novel viral antigen-specific T cell therapy for COVID-19 and potential future pandemics.

The a priori T cell repertoire and immune response against SARS-CoV-2 viral antigens may explain the varying clinical course and prognosis of patients having a mild COVID-19 infection as opposed to those developing more fulminant multisystem organ failure and associated mortality. Using a novel SARS-Cov-2-specific artificial antigen presenting cell (aAPC), coupled with a rapid expansion protocol (REP) as practiced in tumor infiltrating lymphocytes (TIL) therapy, we generate an immune catalytic quantity of Virus Induced Lymphocytes (VIL). Using T cell receptor (TCR)-specific aAPCs carrying co-stimulatory molecules and major histocompatibility complex (MHC) class-I immunodominant SARS-CoV-2 peptide-pentamer complexes, we expand virus-specific VIL derived from peripheral blood mononuclear cells (PBMC) of convalescent COVID-19 patients up to 1000-fold. This is achieved in a clinically relevant 7-day vein-to-vein time-course as a potential adoptive cell therapy (ACT) for COVID-19. We also evaluate this approach for other viral pathogens using Cytomegalovirus (CMV)-specific VIL from donors as a control. Rapidly expanded VIL are enriched in virus antigen-specificity and show an activated, polyfunctional cytokine profile and T effector memory phenotype which may contribute to a robust immune response. Virus-specific T cells can also be delivered allogeneically via MHC-typing and patient human leukocyte antigen (HLA)-matching to provide pragmatic treatment in a large-scale therapeutic setting. These data suggest that VIL may represent a novel therapeutic option that warrants further clinical investigation in the armamentarium against COVID-19 and other possible future pandemics.

Neem leaf glycoprotein directs T-bet-associated type 1 immune commitment.

Neem leaf glycoprotein (NLGP)-mediated immune activation and associated immune polarization was studied. NLGP-induced activation is reflected in upregulation of early activation marker CD69 on lymphocytes, monocytes, and dendritic cells. Activation is also denoted by CD45RO enhancement, with a decrease in CD45RA phenotype and CD62L (L-selectin). NLGP-activated T cells secrete greater amount of signature T-helper (Th)1 cytokines interferon-gamma and a lower amount of the Th2 cytokine interleukin (IL)-4. Similar type 1 directiveness is also observed in antigen-presenting monocytes and dendritic cells by upregulation of IL-12, tumor necrosis factor -alpha and downregulation of IL-10. Creation of the type 1 microenvironment is also assisted by NLGP-induced downregulation of FoxP3(+) T-Reg cells. A type 1-specific transcription factor, T-bet, is upregulated in circulating immune cells after their stimulation with NLGP. In the creation of type 1 immune network, increased phosphorylation of STAT1 and STAT4 with decreased phosphorylation of STAT3 might have significance. We conclude that NLGP may be effective in maintaining normal immune homeostasis by upregulating type 1 response in immunosuppressed hosts, which may have significant role in the induction of host protective antitumor functions.

Bacterial exotoxins downregulate cathelicidin (hCAP-18/LL-37) and human beta-defensin 1 (HBD-1) expression in the intestinal epithelial cells.

Cathelicidin (hCAP-18/LL-37) and beta-defensin 1 (HBD-1) are human antimicrobial peptides (AMPs) with high basal expression levels, which form the first line of host defence against infections over the epithelial surfaces. The antimicrobial functions owe to their direct microbicidal effects as well as the immunomodulatory role. Pathogenic microorganisms have developed multiple modalities including transcriptional repression to combat this arm of the host immune response. The precise mechanisms and the pathogen-derived molecules responsible for transcriptional downregulation remain unknown. Here, we have shown that enteric pathogens suppress LL-37 and HBD-1 expression in the intestinal epithelial cells (IECs) with Vibrio cholerae and enterotoxigenic Escherichia coli (ETEC) exerting the most dramatic effects. Cholera toxin (CT) and labile toxin (LT), the major virulence proteins of V. cholerae and ETEC, respectively, are predominantly responsible for these effects, both in vitro and in vivo. CT transcriptionally downregulates the AMPs by activating several intracellular signalling pathways involving protein kinase A (PKA), ERK MAPKinase and Cox-2 downstream of cAMP accumulation and inducible cAMP early repressor (ICER) may mediate this role of CT, at least in part. This is the first report to show transcriptional repression of the AMPs through the activation of cellular signal transduction pathways by well-known virulence proteins of pathogenic microorganisms.

Dysregulation in immune functions is reflected in tumor cell cytotoxicity by peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients.

We assessed the immunological status of stage III and IV head and neck squamous cell carcinoma (HNSCC) patients and age-matched healthy individuals. In HNSCC patients, the total leukocyte count was lower and the proliferating ability of PBMCs against phytohemagglutinin (PHA) was significantly downregulated. These cells showed lower expression of the early activation marker CD69. Within this PBMC population, the proportion of CD4+, CD8+ T cells, CD3- CD56+, CD16+ NK cells and CD3+ CD56+ NK-T cells was seriously downregulated. However, the proportion of CD4+ CD25+ Foxp3+ regulatory T cells having suppressor function was upregulated. Other immune cells, like CD14+ monocytes/macrophages and CD20+ B cells, were also fewer in number, although this difference was not statistically significant. Assessment of the cytokine secretory status of PBMCs revealed suppressed levels of Th1 cytokines (IFN-gamma, IL-12 and TNF-alpha) and elevated secretion of Th2 cytokines (IL-4 and IL-10) for HNSCC PBMCs whereas just the opposite was seen for PBMCs from healthy individuals. Dysregulation in the profile of immunocompetent cells and cytokine secretion was reflected in the suppressed cytotoxic function of HNSCC PBMCs, as tested on KB (oral cancer), MCF7 (breast cancer), COLO205 (colon cancer), Jurkat (T cell leukemia), K562 (erythroleukemia) and U937 (monocytic lymphoma) cell lines. The observed decreased cytotoxicity of HNSCC PBMCs may be due to the downregulated expression of cytotoxic molecules (perforin, granzymeB and FasL) in HNSCC PBMCs. Assessment of the extent of immune dysfunction might help design immunotherapeutic protocols by incorporating any agent having immunomodulatory function.

Neem leaf glycoprotein restores the impaired chemotactic activity of peripheral blood mononuclear cells from head and neck squamous cell carcinoma patients by maintaining CXCR3/CXCL10 balance.

Interaction between CXCL10 and CXCR3 is dysregulated in head and neck squamous cell carcinoma (HNSCC) and hampers chemotaxis of cytotoxic cells at tumor site. In continuation of the demonstration of significant immunomodulatory effects of neem leaf preparation (NLP), the active ingredient of NLP is characterized as a glycoprotein (NLGP). NLGP is responsible for in vivo immunomodulation to restrict the growth of mice tumors. Effect of NLGP in rectification of the dysregulated IFN gamma dependent chemokine and its receptor CXCR3 splice variants was investigated. Upregulated expression of CXCR3B in HNSCC-PBMC were downregulated following in vitro NLGP treatment. Unchanged expression of CXCR3A+B by NLGP with downregulation of the CXCR3B indirectly suggests the upregulation of the CXCR3A, responsible for cellular migration. However, stimulation of healthy-PBMC with NLGP maintains physiological homeostasis of CXCL10 and increases IFN gamma secretion. The suppressed chemotaxis of HNSCC-PBMC could be restored either by in vitro treatment with NLGP or during use of NLGP stimulated PBMC supernatant as a chemoattractant. Neutralization studies confirmed that the chemoattraction process is guided by both receptor (CXCR3A) and its ligand (CXCL10). Neutralization of the IFN gamma in PBMC culture in presence of NLGP unexpectedly increases the intracellular release of CXCL10, suggesting the NLGP mediated IFN gamma independent release of CXCL10. Interestingly, downregulation of the CXCL10 release was detected after IFN gamma neutralization in absence of NLGP and IFN gamma receptor neutralization in presence of NLGP. Efficacy of NLGP in restoration of the dysregulation of the chemokine signaling may be utilized to design new immunotherapeutic protocol.

PI3Kδ hyper-activation promotes development of B cells that exacerbate Streptococcus pneumoniae infection in an antibody-independent manner.

Streptococcus pneumoniae is a major cause of pneumonia and a leading cause of death world-wide. Antibody-mediated immune responses can confer protection against repeated exposure to S. pneumoniae, yet vaccines offer only partial protection. Patients with Activated PI3Kδ Syndrome (APDS) are highly susceptible to S. pneumoniae. We generated a conditional knock-in mouse model of this disease and identify a CD19+B220- B cell subset that is induced by PI3Kδ signaling, resides in the lungs, and is correlated with increased susceptibility to S. pneumoniae during early phases of infection via an antibody-independent mechanism. We show that an inhaled PI3Kδ inhibitor improves survival rates following S. pneumoniae infection in wild-type mice and in mice with activated PI3Kδ. These results suggest that a subset of B cells in the lung can promote the severity of S. pneumoniae infection, representing a potential therapeutic target.

Severe asthma in humans and mouse model suggests a CXCL10 signature underlies corticosteroid-resistant Th1 bias.

We previously showed that Th1/type 1 inflammation marked by increased IFN-γ levels in the airways can be appreciated in 50% of patients with severe asthma, despite high dose corticosteroid (CS) treatment. We hypothesized that a downstream target of IFN-γ, CXCL10, which recruits Th1 cells via the cognate receptor CXCR3, is an important contributor to Th1high asthma and CS unresponsiveness. We show high levels of CXCL10 mRNA closely associated with IFNG levels in the BAL cells of 50% of severe asthmatics and also in the airways of mice subjected to a severe asthma model, both in the context of high-dose CS treatment. The inability of CS to dampen IFNG or CXCL10 expression was not because of impaired nuclear translocation of the glucocorticoid receptor (GR) or its transactivational functions. Rather, in the presence of CS and IFN-γ, STAT1 and GR were recruited on critical regulatory elements in the endogenous CXCL10 promoter in monocytes, albeit without any abatement of CXCL10 gene expression. High CXCL10 gene expression was also associated with a mast cell signature in both humans and mice, CXCR3 being also expressed by mast cells. These findings suggest that the IFN-γ-CXCL10 axis plays a central role in persistent type 1 inflammation that may be facilitated by CS therapy through GR-STAT1 cooperation converging on the CXCL10 promoter.

IRF5 distinguishes severe asthma in humans and drives Th1 phenotype and airway hyperreactivity in mice.

Severe asthma (SA) is a significant problem both clinically and economically, given its poor response to corticosteroids (CS). We recently reported a complex type 1-dominated (IFN-γ-dominated) immune response in more than 50% of severe asthmatics despite high-dose CS treatment. Also, IFN-γ was found to be critical for increased airway hyperreactivity (AHR) in our model of SA. The transcription factor IRF5 expressed in M1 macrophages can induce a Th1/Th17 response in cocultured human T cells. Here we show markedly higher expression of IRF5 in bronchoalveolar lavage (BAL) cells of severe asthmatics as compared with that in cells from milder asthmatics or healthy controls. Using our SA mouse model, we demonstrate that lack of IRF5 in lymph node migratory DCs severely limits their ability to stimulate the generation of IFN-γ- and IL-17-producing CD4+ T cells and IRF5-/- mice subjected to the SA model displayed significantly lower IFN-γ and IL-17 responses, albeit showing a reciprocal increase in Th2 response. However, the absence of IRF5 rendered the mice responsive to CS with suppression of the heightened Th2 response. These data support the notion that IRF5 inhibition in combination with CS may be a viable approach to manage disease in a subset of severe asthmatics.

The mito-DAMP cardiolipin blocks IL-10 production causing persistent inflammation during bacterial pneumonia.

Bacterial pneumonia is a significant healthcare burden worldwide. Failure to resolve inflammation after infection precipitates lung injury and an increase in morbidity and mortality. Gram-negative bacteria are common in pneumonia and increased levels of the mito-damage-associated molecular pattern (DAMP) cardiolipin can be detected in the lungs. Here we show that mice infected with Klebsiella pneumoniae develop lung injury with accumulation of cardiolipin. Cardiolipin inhibits resolution of inflammation by suppressing production of anti-inflammatory IL-10 by lung CD11b+Ly6GintLy6CloF4/80+ cells. Cardiolipin induces PPARγ SUMOylation, which causes recruitment of a repressive NCOR/HDAC3 complex to the IL-10 promoter, but not the TNF promoter, thereby tipping the balance towards inflammation rather than resolution. Inhibition of HDAC activity by sodium butyrate enhances recruitment of acetylated histone 3 to the IL-10 promoter and increases the concentration of IL-10 in the lungs. These findings identify a mechanism of persistent inflammation during pneumonia and indicate the potential of HDAC inhibition as a therapy.

Mitochondrial H2O2 in Lung Antigen-Presenting Cells Blocks NF-κB Activation to Prevent Unwarranted Immune Activation.

Inhalation of environmental antigens such as allergens does not always induce inflammation in the respiratory tract. While antigen-presenting cells (APCs), including dendritic cells and macrophages, take up inhaled antigens, the cell-intrinsic molecular mechanisms that prevent an inflammatory response during this process, such as activation of the transcription factor NF-κB, are not well understood. Here, we show that the nuclear receptor PPARγ plays a critical role in blocking NF-κB activation in response to inhaled antigens to preserve immune tolerance. Tolerance induction promoted mitochondrial respiration, generation of H2O2, and suppression of NF-κB activation in WT, but not PPARγ-deficient, APCs. Forced restoration of H2O2 in PPARγ-deficient cells suppressed IκBα degradation and NF-κB activation. Conversely, scavenging reactive oxygen species from mitochondria promoted IκBα degradation with loss of regulatory and promotion of inflammatory T cell responses in vivo. Thus, communication between PPARγ and the mitochondria maintains immune quiescence in the airways.

Challenges and future in vaccines, drug development, and immunomodulatory therapy.

Pulmonary diseases and infections are among the top contributors to human morbidity and mortality worldwide, and despite the successful history of vaccines and antimicrobial therapeutics, infectious disease still presents a significant threat to human health. Effective vaccines are frequently unavailable in developing countries, and successful vaccines have yet to be developed for major global maladies, such as tuberculosis. Furthermore, antibiotic resistance poses a growing threat to human health. The "Challenges and Future in Vaccines, Drug Development, and Immunomodulatory Therapy" session of the 2013 Pittsburgh International Lung Conference highlighted several recent and current studies related to treatment and prevention of antibiotic-resistant bacterial infections, highly pathogenic influenza, respiratory syncytial virus, and tuberculosis. Research presented here focused on novel antimicrobial therapies, new vaccines that are either in development or currently in clinical trials, and the potential for immunomodulatory therapies. These studies are making important contributions to the areas of microbiology, virology, and immunology related to pulmonary diseases and infections and are paving the way for improvements in the efficacy of vaccines and antimicrobials.