Effect of monoclonal antibody therapy on the endogenous SARS-CoV-2 antibody response.

Monoclonal antibody treatment of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection has been widely implemented. Effects of treatment on the endogenous primary humoral response to the virus are unknown. A retrospective cohort study performed at a Veterans Health Administration medical center compared serologic responses of treated and untreated COVID-19 patients at high risk for severe outcomes. Three anti-viral spike protein IgG monoclonal treatments were used during the study period, 1) bamlanivimab, 2) casirivimab with imdevimab, and 3) bamlanivimab with etesevimab. Data were analyzed at acute (0-9 days), seroconversion (10-19 days), and maximum antibody (20-39 days) stages. SARS-Cov-2 infection induced a dynamic primary humoral response with anti-spike IgM and anti-nucleocapsid IgG seroconversion occurring after 9 days with maximum serologic indices achieved by 20-39 days. All monoclonal antibody treatments suppressed the endogenous anti-spike IgM response by 85-90% with minor effect on the anti-nucleocapsid response. Thus, passive immunization therapy may cause immunologic interference.

Role of CC chemokine receptor 4 in natural killer cell activation during acute cigarette smoke exposure.

Cigarette smoke (CS)-induced lung injury involves innate immune responses. The activation of innate effector cells is thought to require cross talk with dendritic cells (DCs) and macrophages, but the mediators of interaction are unknown. One candidate, CC chemokine receptor 4 (CCR4), is expressed by innate and adaptive effector cells, and its ligands are produced by DCs and macrophages. Using flow cytometry and confocal microscopy, we defined innate responses of lung myeloid DCs, macrophages, and conventional natural killer (NK) cells in mice exposed to CS over 4 days and examined the contribution of CCR4 using CCR4 knockout (CCR4(-/-)) mice. CS affected populations differently, causing an increase in F4/80(+) macrophages, a reduction in parenchymal CD11c(+)CD11b(+)CD103(-) DCs, but no effect on mucosal CD11c(+)CD11b(-)CD103(+) DCs. CS also induced a population of primed/activated CD69(+) NK cells and bronchoepithelial expression of the stress-related NKG2D receptor-activating protein, retinoic acid early transcript 1. CS-exposed CCR4(-/-) mice were similar to controls regarding effects on DCs and macrophages but displayed substantially impaired NK priming/activation and reduced expression of transcripts for interferon gamma, CXCL10, and retinoic acid early transcript 1. Quantitative confocal microscopy revealed that lungs of CS-exposed CCR4(-/-) mice had significantly reduced contacts of NK cells with CD11c(+) cells. These findings demonstrate that acute CS exposure elicits NK cell responses and suggest that CCR4 promotes NK cell priming/activation by mediating contacts with sentinel cells in the lung.

The host environment is responsible for aging-related functional NK cell deficiency.

NK cells play an important role in immunity against infection and tumors. Aging-related functional NK cell deficiency is well documented in humans and mice. However, the mechanism for this is poorly understood. Using an adoptive transfer approach in mice, we found that NK cells from both young and aged mice responded vigorously to priming by pathogen-derived products after being cotransferred into young mice. In contrast, NK cells from young mice responded poorly to priming by pathogen-derived products after being transferred to aged mice. In addition to defects in NK cell priming, maturation of NK cells under steady-state conditions is also impaired in aged mice, resulting in a decreased proportion of CD27(-) mature NK cells. We found that bone marrow from young and aged mice gave rise to CD27(-) mature NK cells similarly in young mixed bone marrow chimeric mice. Furthermore, by using a novel bone marrow transfer approach without irradiation, we found that after being transferred to aged mice, bone marrow from young mice gave rise to NK cells with maturation defects. Finally, we found that aging-related functional NK cell deficiency was completely reversed by injecting soluble IL-15/IL-15Rα complexes. In contrast, blockade of IL-10 signaling, which broadly augments inflammatory responses to pathogen-derived products, had little effect on aging-related defects in NK cell priming. These data demonstrate that the aged host environment is responsible for aging-related functional NK cell deficiency. Additionally, our data suggest that IL-15 receptor agonists may be useful tools in treating aging-related functional NK cell deficiency.

Cutting edge: Central memory CD8 T cells in aged mice are virtual memory cells.

The number of memory phenotype CD8 T cells increases dramatically with aging in both humans and mice. However, the mechanism for this is unknown. The prevailing hypothesis is that memory T cells accumulate with aging as a result of lifelong antigenic stimulation. However, data supporting this supposition are lacking. In this study, we demonstrate that central memory CD8 T cells, which represent a large majority of memory CD8 T cells in aged mice, are not memory cells that develop in response to antigenic stimulation but are virtual memory cells that develop without antigenic stimulation. In addition to phenotypic evidence, we show that accumulation of central memory CD8 T cells is independent of CD4 T cells, CCR5, and CXCR3, all of which are known to be essential for Ag-driven development of central memory CD8 T cells. Thus, this study reveals a novel mechanism for aging-related changes in CD8 T cells.

Lung CD8+ T cells in COPD have increased expression of bacterial TLRs.

BACKGROUND: Toll-like receptors (TLRs) on T cells can modulate their responses, however, the extent and significance of TLR expression by lung T cells, NK cells, or NKT cells in chronic obstructive pulmonary disease (COPD) is unknown. METHODS: Lung tissue collected from clinically-indicated resections (n = 34) was used either: (a) to compare the expression of TLR1, TLR2, TLR2/1, TLR3, TLR4, TLR5, TLR6 and TLR9 on lung CD8+ T cells, CD4+ T cells, NK cells and NKT cells from smokers with or without COPD; or (b) to isolate CD8+ T cells for culture with anti-CD3ε without or with various TLR ligands. We measured protein expression of IFN-γ, TNF-α, IL-13, perforin, granzyme A, granzyme B, soluble FasL, CCL2, CCL3, CCL4, CCL5, CCL11, and CXCL9 in supernatants. RESULTS: All the lung subsets analyzed demonstrated low levels of specific TLR expression, but the percentage of CD8+ T cells expressing TLR1, TLR2, TLR4, TLR6 and TLR2/1 was significantly increased in COPD subjects relative to those without COPD. In contrast, from the same subjects, only TLR2/1 and TLR2 on lung CD4+ T cells and CD8+ NKT cells, respectively, showed a significant increase in COPD and there was no difference in TLR expression on lung CD56+ NK cells. Production of the Tc1 cytokines IFN-γ and TNF-α by lung CD8+ T cells were significantly increased via co-stimulation by Pam3CSK4, a specific TLR2/1 ligand, but not by other agonists. Furthermore, this increase in cytokine production was specific to lung CD8+ T cells from patients with COPD as compared to lung CD8+ T cells from smokers without COPD. CONCLUSIONS: These data suggest that as lung function worsens in COPD, the auto-aggressive behavior of lung CD8+ T cells could increase in response to microbial TLR ligands, specifically ligands against TLR2/1.

Smoking decreases the response of human lung macrophages to double-stranded RNA by reducing TLR3 expression.

BACKGROUND: Cigarette smoking is associated with increased frequency and duration of viral respiratory infections, but the underlying mechanisms are incompletely defined. We investigated whether smoking reduces expression by human lung macrophages (Mø) of receptors for viral nucleic acids and, if so, the effect on CXCL10 production. METHODS: We collected alveolar macrophages (AMø) by bronchoalveolar lavage of radiographically-normal lungs of subjects undergoing bronchoscopies for solitary nodules (n = 16) and of volunteers who were current or former smokers (n = 7) or never-smokers (n = 13). We measured expression of mRNA transcripts for viral nucleic acid receptors by real-time PCR in those AMø and in the human Mø cell line THP-1 following phorbol myristate acetate/vitamin D3 differentiation and exposure to cigarette smoke extract, and determined TLR3 protein expression using flow cytometry and immunohistochemistry. We also used flow cytometry to examine TLR3 expression in total lung Mø from subjects undergoing clinically-indicated lung resections (n = 25). Of these, seven had normal FEV1 and FEV1/FVC ratio (three former smokers, four current smokers); the remaining 18 subjects (14 former smokers; four current smokers) had COPD of GOLD stages I-IV. We measured AMø production of CXCL10 in response to stimulation with the dsRNA analogue poly(I:C) using Luminex assay. RESULTS: Relative to AMø of never-smokers, AMø of smokers demonstrated reduced protein expression of TLR3 and decreased mRNA for TLR3 but not TLR7, TLR8, TLR9, RIG-I, MDA-5 or PKR. Identical changes in TLR3 gene expression were induced in differentiated THP-1 cells exposed to cigarette smoke-extract in vitro for 4 hours. Among total lung Mø, the percentage of TLR3-positive cells correlated inversely with active smoking but not with COPD diagnosis, FEV1% predicted, sex, age or pack-years. Compared to AMø of never-smokers, poly(I:C)-stimulated production of CXCL10 was significantly reduced in AMø of smokers. CONCLUSIONS: Active smoking, independent of COPD stage or smoking duration, reduces both the percent of human lung Mø expressing TLR3, and dsRNA-induced CXCL10 production, without altering other endosomal or cytoplasmic receptors for microbial nucleic acids. This effect provides one possible mechanism for increased frequency and duration of viral lower respiratory tract infections in smokers. TRIAL REGISTRATION: ClinicalTrials.gov NCT00281190, NCT00281203 and NCT00281229.

TLR9 regulates the mycobacteria-elicited pulmonary granulomatous immune response in mice through DC-derived Notch ligand delta-like 4.

TLR9 activation is important for the maintenance of mycobacteria-elicited pulmonary granulomatous responses, hallmarks of protective immune responses following mycobacterial infection. However, the mechanism or mechanisms underlying this effect of TLR9 are not clear. Here, we show that Tlr9-deficient mice challenged with a Mycobacterium antigen display an altered Th17 cytokine profile, decreased accumulation of granuloma-associated myeloid DCs, and profoundly impaired delta-like 4 (dll4) Notch ligand expression. Mechanistic analysis revealed that WT bone marrow-derived DCs but not macrophages promoted the differentiation of Th17 cells from bacillus Calmette-Guérin-challenged (BCG-challenged) lung CD4+ T cells. Both lung and bone marrow DCs isolated from Tlr9-deficient mice inoculated with Mycobacterium antigen expressed lower levels of dll4 Notch ligand than the same cells isolated from WT mice. Passively immunizing WT mice with neutralizing antibodies specific for dll4 during granuloma formation resulted in larger granulomas and lower levels of Th17-related cytokines. In addition, dll4 specifically regulated Th17 activation in vitro. Together, these results suggest dll4 plays an important role in promoting Th17 effector activity during a mycobacterial challenge. Furthermore, TLR9 seems to be required for optimal dll4 expression and the regulation of Mycobacterium antigen-elicited granuloma formation in mice.

CC chemokine receptor 4 contributes to innate NK and chronic stage T helper cell recall responses during Mycobacterium bovis infection.

Cysteine-cysteinyl chemokine receptor 4 (CCR4) is expressed by a variety of T-cell subsets and leukocytes. This study examined the participation of CCR4 in response to pulmonary infection with Mycobacterium bovis Bacille-Calmette-Guerin (BCG). Constitutive and induced CCR4 agonist expression was detected among large mononuclear cells. The course of infection and mobilization of effector cell populations were then analyzed in CCR4 knockout (CCR4(-/-)) mice. Compared with controls, CCR4(-/-) mice displayed delayed innate stage (<2 weeks) bacterial clearance and reduced late stage inflammation. Innate impairment was associated with reduced natural killer cell activation. In the adaptive phase, CCR4(-/-) mice generated effector T cells in draining lymph nodes and accumulated effector T cells in lungs, which resulted in normal adaptive stage bacterial elimination at 2 to 4 weeks. However, during the late stage, CCR4(-/-) mice had reduced interferonγ+CD4(+)α/ß+ (Th1) and interleukin (IL)-17+CD4(+)α/ß+ (Th17) T helper cells in lungs. In contrast, IL-17+ γ/δ T cells in lungs were unaffected. When challenged with mycobacterial antigen- (Ag-) Ag-coated beads to elicit a recall granulomatous response, CCR4(-/-) mice displayed abrogated recall granuloma formation and reduced interferon γ+ Th1 cells. These findings indicate that CCR4 supports innate natural killer cell activation and sustains later CD4(+) Th effector/memory antimycobacterial responses in the lung but is redundant in the early adaptive elimination phase.

IL-4 acts as a potent stimulator of IFN-γ expression in CD8+ T cells through STAT6-dependent and independent induction of Eomesodermin and T-bet.

CD8+ T cell synthesis of IFN-γ is an important component of the CD8+ T cell immune response. In short-term cultures of murine pan-T cells, we found that IL-4 was the principal cytokine responsible for driving IFN-γ synthesis by CD3/CD28-activated CD8+ T cells. IL-4 was able to induce low levels of IFN-γ mRNA in CD8+ T cells even in the absence of CD3/CD28 engagement, although concomitant CD3/CD28 stimulation was necessary for IFN-γ secretion. IL-4 induction of IFN-γ was explained by its ability to induce Eomesodermin and T-bet transcription factors whose expression was further increased by CD3/CD28. Expression of Eomesodermin, T-bet and IFN-γ induced by IL-4 was partially dependent upon activation of MAPK and PI3K but independent of the canonical IL-4-activated transcription factor, STAT6. In contrast, expression of IFN-γ induced by IL-4/CD3/CD28 stimulation showed additional dependency upon STAT6 which functions to increase expression of Eomesodermin specifically. These novel findings point to a function for IL-4 as a direct regulator of IFN-γ expression in CD8+ T cells and reveal the molecular mechanisms involved.

Cytotoxic potential of lung CD8(+) T cells increases with chronic obstructive pulmonary disease severity and with in vitro stimulation by IL-18 or IL-15.

Lung CD8(+) T cells might contribute to progression of chronic obstructive pulmonary disease (COPD) indirectly via IFN-gamma production or directly via cytolysis, but evidence for either mechanism is largely circumstantial. To gain insights into these potential mechanisms, we analyzed clinically indicated lung resections from three human cohorts, correlating findings with spirometrically defined disease severity. Expression by lung CD8(+) T cells of IL-18R and CD69 correlated with severity, as did mRNA transcripts for perforin and granzyme B, but not Fas ligand. These correlations persisted after correction for age, smoking history, presence of lung cancer, recent respiratory infection, or inhaled corticosteroid use. Analysis of transcripts for killer cell lectin-like receptor G1, IL-7R, and CD57 implied that lung CD8(+) T cells in COPD do not belong to the terminally differentiated effector populations associated with chronic infections or extreme age. In vitro stimulation of lung CD8(+) T cells with IL-18 plus IL-12 markedly increased production of IFN-gamma and TNF-alpha, whereas IL-15 stimulation induced increased intracellular perforin expression. Both IL-15 and IL-18 protein expression could be measured in whole lung tissue homogenates, but neither correlated in concentration with spirometric severity. Although lung CD8(+) T cell expression of mRNA for both T-box transcription factor expressed in T cells and GATA-binding protein 3 (but not retinoic acid receptor-related orphan receptor gamma or alpha) increased with spirometric severity, stimulation of lung CD8(+) T cells via CD3epsilon-induced secretion of IFN-gamma, TNF-alpha, and GM-CSF, but not IL-5, IL-13, and IL-17A. These findings suggest that the production of proinflammatory cytokines and cytotoxic molecules by lung-resident CD8(+) T cells contributes to COPD pathogenesis.

SARS-CoV-2 Anti-Spike IgG Antibody and ACE2 Receptor Binding Inhibition Levels among Breakthrough Stage Veteran Patients.

SARS-CoV-2 mRNA vaccines have been critical to curbing pandemic COVID-19; however, a major shortcoming has been the inability to assess levels of protection after vaccination. This study assessed serologic status of breakthrough infections in vaccinated patients at a Veterans Administration medical center from June through December 2021 during a SARS-CoV-2 delta variant wave. Breakthrough occurred mostly beyond 150 days after two-dose vaccination with a mean of 239 days. Anti-SARS-CoV-2 spike (S) IgG levels were low at 0 to 2 days postsymptoms but increased in subjects presenting thereafter. Population measurements of anti-S IgG and angiotensin converting enzyme-2 receptor (ACE2-R) binding inhibition among uninfected, vaccinated patients suggested immune decay occurred after 150 days with 62% having anti-S IgG levels at or below 1,000 AU comparable with breakthrough patients at 0 to 2 days postsymptom onset. In contrast, vaccination after resolved infection conferred robust enduring anti-S IgG levels (5,000 to >50,000 AU) with >90% ACE2-R binding inhibition. However, monoclonal antibody (MAb)-treated patients did not benefit from their prior infection suggesting impaired establishment of B cell memory. Analysis of boosted patients confirmed the benefit of a third vaccine dose with most having anti-S IgG levels above 5,000 AU with >90% ACE2-R binding inhibition, but a subset had levels <5,000 AU. Anti-S IgG levels >5,000 AU were associated with >90% ACE2-R binding inhibition and no documented breakthrough infections, whereas levels falling below 5,000 AU and approaching 1,000 AU were associated with breakthrough infections. Thus, quantitative antibody measurements may provide a means to guide vaccination intervals for the individual. IMPORTANCE Currently, clinicians have no guidance for the serologic assessment of SARS-Cov-2 postvaccination status regarding protection and risk of infection. Vaccination and boosters are administered blindly without evaluation of need or outcome at the individual level. The recent development of automated quantitative assays for anti-SARS-CoV-2 spike protein IgG antibodies permits accurate measurement of humoral immunity in standardized units. Clinical studies, such as reported here, will help establish protective antibody levels allowing identification and targeted management of poor vaccine responders and vaccinated subjects undergoing immune decay.

EAE mediated by a non-IFN-γ/non-IL-17 pathway.

Previous studies have shown that EAE can be elicited by the adoptive transfer of either IFN-γ-producing (Th1) or IL-17-producing (Th17) myelin-specific CD4(+) T-cell lines. Paradoxically, mice deficient in either IFN-γ or IL-17 remain susceptible to EAE following immunization with myelin antigens in CFA. These observations raise questions about the redundancy of IFN-γ and IL-17 in autoimmune demyelinating disease mediated by a diverse, polyclonal population of autoreactive T cells. In this study, we show that an atypical form of EAE, induced in C57BL/6 mice by the adoptive transfer of IFN-γ-deficient effector T cells, required IL-17 signaling for the development of brainstem infiltrates. In contrast, classical EAE, characterized by predominant spinal cord inflammation, occurred in the combined absence of IFN-γ and IL-17 signaling, but was dependent on GM-CSF and CXCR2. Our findings contribute to a growing body of data, indicating that individual cytokines vary in their importance across different models of CNS autoimmunity.

CD11c+ cells are required to prevent progression from local acute lung injury to multiple organ failure and death.

To investigate the role of CD11c(+) cells in endotoxin-induced acute lung injury, wild-type or CD11c-diphtheria toxin receptor transgenic mice were treated with intraperitoneal diphtheria toxin (5 ng/g b.wt.) in the presence or absence of intratracheal lipopolysaccharide (51 microg). Lipopolysaccharide treatment resulted in 100% mortality in CD11c-depleted animals but not in control animals. Analysis of local lung tissue revealed no differences in acute lung injury severity; however, analysis of distal tissues revealed severe damage and necrosis to multiple organs (liver, spleen, and kidneys) in CD11c-diphtheria toxin receptor mice but not in wild-type mice. In addition, dramatic increases in systemic levels of liver enzymes (alanine aminotransferase, 657 U/L, aspartate aminotransferase, 1401 U/L), blood urea (53 mg/dl), and 8-iso-prostaglandin F(2alpha), a marker of oxidative stress (350 pg/ml), were observed. These data demonstrate that CD11c(+) cells play a critical role in protecting the organs from systemic injury caused by a pulmonary endotoxin challenge.

Seroprevalence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection among Veterans Affairs healthcare system employees suggests higher risk of infection when exposed to SARS-CoV-2 outside the work environment.

OBJECTIVE: The seroprevalence of severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) IgG antibody was evaluated among employees of a Veterans Affairs healthcare system to assess potential risk factors for transmission and infection. METHODS: All employees were invited to participate in a questionnaire and serological survey to detect antibodies to SARS-CoV-2 as part of a facility-wide quality improvement and infection prevention initiative regardless of clinical or nonclinical duties. The initiative was conducted from June 8 to July 8, 2020. RESULTS: Of the 2,900 employees, 51% participated in the study, revealing a positive SARS-CoV-2 seroprevalence of 4.9% (72 of 1,476; 95% CI, 3.8%-6.1%). There were no statistically significant differences in the presence of antibody based on gender, age, frontline worker status, job title, performance of aerosol-generating procedures, or exposure to known patients with coronavirus infectious disease 2019 (COVID-19) within the hospital. Employees who reported exposure to a known COVID-19 case outside work had a significantly higher seroprevalence at 14.8% (23 of 155) compared to those who did not 3.7% (48 of 1,296; OR, 4.53; 95% CI, 2.67-7.68; P < .0001). Notably, 29% of seropositive employees reported no history of symptoms for SARS-CoV-2 infection. CONCLUSIONS: The seroprevalence of SARS-CoV-2 among employees was not significantly different among those who provided direct patient care and those who did not, suggesting that facility-wide infection control measures were effective. Employees who reported direct personal contact with COVID-19-positive persons outside work were more likely to have SARS-CoV-2 antibodies. Employee exposure to SARS-CoV-2 outside work may introduce infection into hospitals.

Mononuclear phagocyte-derived IL-10 suppresses the innate IL-12/IFN-gamma axis in lung-challenged aged mice.

Previously, we reported that IL-10-producing mononuclear phagocytes increase in lungs of aged mice, causing impaired innate cytokine expression. Since dendritic cells (DCs) contribute to innate NK cell and adaptive T cell immunity, we tested the hypothesis that age-related IL-10 might influence DC function with effects on NK and T cell activation. The results showed that DC recruitment to sites of lung inflammation was normal in aged mice (>20 mo). However, IFN-gamma-producing NK cells in LPS-challenged lungs were decreased in aged as compared with young mice, which was associated with increased IL-10(+)CD11b(+)Gr-1(low)CD11c(-) cells consistent with mononuclear phagocytes. In vivo or in vitro blockade of IL-10 signaling restored IFN-gamma-producing NK cells. This restoration was reversed by IL-12 neutralization, indicating that IL-10 suppressed sources of IL-12 in aged mice. To probe DC function in adaptive immunity, we transferred young naive OVA-specific TCR transgenic T cells to old mice. Following challenge with OVA plus LPS, Ag presentation in the context of MHC-I and MHC-II occurred with similar kinetics and intensity in draining lymph nodes of young and old recipients as measured by proliferation. Despite this, aged hosts displayed impaired induction of IFN-gamma(+)CD4(+), but not IFN-gamma(+)CD8(+), effector T cells. Blockade of IL-10 signaling reversed age-associated defects. These studies indicate that the innate IL-12/IFN-gamma axis is not intrinsically defective in lungs of aged mice, but is rather suppressed by enhanced production of mononuclear phagocyte-derived IL-10. Our data identify a novel mechanism of age-associated immune deficiency.

Lung dendritic cell expression of maturation molecules increases with worsening chronic obstructive pulmonary disease.

RATIONALE: Dendritic cells (DCs) have not been well studied in chronic obstructive pulmonary disease (COPD), yet their integral role in activating and differentiating T cells makes them potential participants in COPD pathogenesis. OBJECTIVES: To determine the expression of maturation molecules by individual DC subsets in relationship to COPD stage and to expression of the acute activation marker CD69 by lung CD4(+) T cells. METHODS: We nonenzymatically released lung leukocytes from human surgical specimens (n = 42) and used flow cytometry to identify three DC subsets (mDC1, mDC2, and pDC) and to measure their expression of three costimulatory molecules (CD40, CD80 and CD86) and of CD83, the definitive marker of DC maturation. Spearman nonparametric correlation analysis was used to identify significant correlations between expression of DC maturation molecules and COPD severity. MEASUREMENTS AND MAIN RESULTS: Expression of CD40 by mDC1 and mDC2 and of CD86 by mDC2 was high regardless of GOLD stage, but CD80 and CD83 on these two DC subsets increased with disease progression. pDC also showed significant increases in expression of CD40 and CD80. Expression of all but one of the DC molecules that increased with COPD severity also correlated with CD69 expression on lung CD4(+) T cells from the same patients, with the exception of CD83 on mDC2. CONCLUSIONS: This cross-sectional study implies that COPD progression is associated with significant increases in costimulatory molecule expression by multiple lung DC subsets. Interactions with lung DCs may contribute to the immunophenotype of CD4(+) T cells in advanced COPD. Clinical trial registered with www.clinicaltrials.gov (NCT00281229).

Toll-like receptor 9 activation is a key mechanism for the maintenance of chronic lung inflammation.

RATIONALE: Accumulating evidence supports the hypothesis that the continuous host response to a persistent challenge can polarize the cytokine environment toward a Th2 cytokine phenotype, but the mechanisms responsible for this skewing are not clear. OBJECTIVES: We investigated the role of Toll-like receptor 9 (TLR9) in a Th2-driven pulmonary granulomatous response initiated via the embolization of Schistosoma mansoni eggs to the lungs of mice. METHODS: Mice were intravenously injected with S. mansoni eggs. Histological and flow cytometric analysis, cytokine measurement, adoptive transfer of bone marrow (BM)-derived dendritic cells (DCs), and in vitro T-cell treatments with antigen-presenting cells were examined. MEASUREMENTS AND MAIN RESULTS: In comparison to wild-type mice, TLR9(-/-) mice showed increased pulmonary granuloma size, augmented collagen deposition, increased Th2 cytokine phenotype, and impaired accumulation of DCs. BM-derived DCs, but not macrophages, recovered from animals with developed Th2-type lung granulomas promoted the production of type 2 cytokines from CD4(+) T cells. BM-derived DCs from TLR9(-/-) mice induced impaired Th1 cytokine and enhanced Th2 cytokine production by T cells, compared with DCs from WT mice. Macrophages from TLR9(-/-) mice expressed a significantly higher alternatively activated (M2) phenotype characterized by increased "found in inflammatory zone-1" (FIZZ1) and arginase-1 expression. The adoptive transfer of BM-derived DCs from syngeneic WT mice into TLR9(-/-) mice restored the granuloma phenotype seen in WT mice. CONCLUSIONS: These studies suggest that TLR9 plays an important mechanistic role in the maintenance of the pulmonary granulomatous response.

Bone marrow-derived progenitor cells in pulmonary fibrosis.

The origin of fibroblasts in pulmonary fibrosis is assumed to be intrapulmonary, but their extrapulmonary origin and especially derivation from bone marrow (BM) progenitor cells has not been ruled out. To examine this possibility directly, adult mice were durably engrafted with BM isolated from transgenic mice expressing enhanced GFP. Induction of pulmonary fibrosis in such chimera mice by endotracheal bleomycin (BLM) injection caused large numbers of GFP(+) cells to appear in active fibrotic lesions, while only a few GFP(+) cells could be identified in control lungs. Flow-cytometric analysis of lung cells confirmed the BLM-induced increase in GFP(+) cells in chimera mice and revealed a significant increase in GFP(+) cells that also express type I collagen. GFP(+) lung fibroblasts isolated from chimera mice expressed collagen and telomerase reverse transcriptase but not alpha-smooth muscle actin. Treatment of isolated GFP(+) fibroblasts with TGF-beta failed to induce myofibroblast differentiation. Cultured lung fibroblasts expressed the chemokine receptors CXCR4 and CCR7 and responded chemotactically to their cognate ligands, stromal cell-derived factor-1 alpha and secondary lymphoid chemokine, respectively. Thus the collagen-producing lung fibroblasts in pulmonary fibrosis can also be derived from BM progenitor cells.

Increased Foxp3(+) Treg cell activity reduces dendritic cell co-stimulatory molecule expression in aged mice.

As potent suppressors of immune responses to self- and foreign-antigens, Foxp3(+) Treg cells are suspected to be involved in immunosuppression leading to cancer, neurodegeneration and infection. Since ageing is associated with increased incidence of these diseases, we compared Treg activity in blood, lymphoid organs and lungs of young (5-6 months) and old (21-22 months) mice. Both the proportion and absolute number of Foxp3(+) CD4(+) Treg cells increased with age in secondary lymphoid organs but not in blood and lungs as compared to Foxp3(-) CD4(+) T cells. Although numbers of thymic and naïve conventional T and Treg cells decreased with age, Treg cells with memory/effector phenotype increased disproportionately in peripheral lymphoid tissues. In addition, CD40 and CD86 co-stimulatory molecule expression by lymph node dendritic cells was impaired in old mice and could be restored to levels of young mice by inactivating Treg cells with anti-CD25 monoclonal antibodies. These findings have important implications for the understanding of age-related immune dysfunction.

Disruption of Early Tumor Necrosis Factor Alpha Signaling Prevents Classical Activation of Dendritic Cells in Lung-Associated Lymph Nodes and Development of Protective Immunity against Cryptococcal Infection.

UNLABELLED: Anti-tumor necrosis factor alpha (anti-TNF-α) therapies have been increasingly used to treat inflammatory diseases and are associated with increased risk of invasive fungal infections, including Cryptococcus neoformans infection. Using a mouse model of cryptococcal infection, we investigated the mechanism by which disruption of early TNF-α signaling results in the development of nonprotective immunity against C. neoformans We found that transient depletion of TNF-α inhibited pulmonary fungal clearance and enhanced extrapulmonary dissemination of C. neoformans during the adaptive phase of the immune response. Higher fungal burdens in TNF-α-depleted mice were accompanied by markedly impaired Th1 and Th17 responses in the infected lungs. Furthermore, early TNF-α depletion also resulted in disrupted transcriptional initiation of the Th17 polarization program and subsequent upregulation of Th1 genes in CD4(+) T cells in the lung-associated lymph nodes (LALN) of C. neoformans-infected mice. These defects in LALN T cell responses were preceded by a dramatic shift from a classical toward an alternative activation of dendritic cells (DC) in the LALN of TNF-α-depleted mice. Taken together, our results indicate that early TNF-α signaling is required for optimal DC activation, and the initial Th17 response followed by Th1 transcriptional prepolarization of T cells in the LALN, which further drives the development of protective immunity against cryptococcal infection in the lungs. Thus, administration of anti-TNF-α may introduce a particularly greater risk for newly acquired fungal infections that require generation of protective Th1/Th17 responses for their containment and clearance. IMPORTANCE: Increased susceptibility to invasive fungal infections in patients on anti-TNF-α therapies underlines the need for understanding the cellular effects of TNF-α signaling in promoting protective immunity to fungal pathogens. Here, we demonstrate that early TNF-α signaling is required for classical activation and accumulation of DC in LALN of C. neoformans-infected mice. Subsequent transcriptional initiation of Th17 followed by Th1 programming in LALN results in pulmonary accumulation of gamma interferon- and interleukin-17A-producing T cells and effective fungal clearance. All of these crucial steps are severely impaired in mice that undergo anti-TNF-α treatment, consistent with their inability to clear C. neoformans This study identified critical interactions between cells of the innate immune system (DC), the emerging T cell responses, and cytokine networks with a central role for TNF-α which orchestrate the development of the immune protection against cryptococcal infection. This information will be important in aiding development and understanding the potential side effects of immunotherapies.