Dennis M. Lindell

IL-17–Induced Pulmonary Pathogenesis during Respiratory Viral Infection and Exacerbation of Allergic Disease

Severe respiratory syncytial virus (RSV) infections are characterized by airway epithelial cell damage, mucus hypersecretion, and Th2 cytokine production. Less is known about the role of IL-17. We observed increased IL-6 and IL-17 levels in tracheal aspirate samples from severely ill infants with RSV infection. In a mouse model of RSV infection, time-dependent increases in pulmonary IL-6, IL-23, and IL-17 expression were observed. Neutralization of IL-17 during infection and observations from IL-17(-/-) knockout mice resulted in significant inhibition of mucus production during RSV infection. RSV-infected animals treated with anti-IL-17 had reduced inflammation and decreased viral load, compared with control antibody-treated mice. Blocking IL-17 during infection resulted in significantly increased RSV-specific CD8 T cells. Factors associated with CD8 cytotoxic T lymphocytes, T-bet, IFN-γ, eomesodermin, and granzyme B were significantly up-regulated after IL-17 blockade. Additionally, in vitro analyses suggest that IL-17 directly inhibits T-bet, eomesodermin, and IFN-γ in CD8 T cells. The role of IL-17 was also investigated in RSV-induced exacerbation of allergic airway responses, in which neutralization of IL-17 led to a significant decrease in the exacerbated disease, including reduced mucus production and Th2 cytokines, with decreased viral proteins. Taken together, our data demonstrate that IL-17 plays a pathogenic role during RSV infections.

The role of macrophage inflammatory protein-1α/CCL3 in regulation of T cell-mediated immunity to Cryptococcus neoformans infection.

Macrophage inflammatory protein-1α (MIP-1α/CCL3) is a CC chemokine required for optimal recruitment of leukocytes in response to cryptococcal Ags. MIP-1α is expressed in the lungs by day 6 post Cryptococcus neoformans infection and could play a role in the development of cell-mediated immunity. To address this possibility, wild-type (MIP-1α+/+) mice and MIP-1α knockout (MIP-1α−/−) mice were infected intratracheally with a highly virulent strain of C. neoformans (145A). MIP-1α message was detected in the lungs on days 3, 7, and 14 in MIP-1α+/+ mice, but it was undetectable in MIP-1α−/− mice. On day 16, MIP-1α−/− mice had a 7-fold increase in C. neoformans burden in the lungs, but no decrease in pulmonary leukocyte recruitment. MIP-1α+/+ and MIP-1α−/− mice had similar numbers of recruited lymphocytes and monocytes/macrophages. Notably, MIP-1α−/− mice had a significantly greater number of eosinophils. MIP-1α−/− mice had extremely high levels of serum IgE. This switch of immune response to a T2 phenotype was associated with enhanced IL-4 and IL-13 expression in the lungs of MIP-1α−/− mice compared with MIP-1α +/+ mice. Progression of pulmonary cryptococcosis in the presence of nonprotective T2 immunity resulted in profound lung damage in MIP-1α−/− mice (eosinophilic crystal deposition, destruction of lung parenchyma, and pulmonary hemorrhage). Twelve-week survival was dramatically decreased in MIP-1α−/− mice. These studies, together with our previous studies, demonstrate that MIP-1α plays a role in both the afferent (T1/T2 development) and efferent (T1-mediated leukocyte recruitment) phases of cell-mediated immunity to C. neoformans.

The Balance between Plasmacytoid DC versus Conventional DC Determines Pulmonary Immunity to Virus Infections

Background Respiratory syncytial virus (RSV) infects nearly all infants by age 2 and is a leading cause of bronchiolitis. RSV may employ several mechanisms to induce immune dysregulation, including dendritic cell (DC) modulation during the immune response to RSV. Methods and Findings Expansion of cDC and pDC by Flt3L treatment promoted an anti-viral response with reduced pathophysiology characterized by decreased airway hyperreactivity, reduced Th2 cytokines, increased Th1 cytokines, and a reduction in airway inflammation and mucus overexpression. These protective aspects of DC expansion could be completely reversed by depleting pDCs during the RSV infection. Expansion of DCs by Flt3L treatment enhanced in CD8+ T cell responses, which was reversed by depletion of pDC. Conclusions These results indicate that a balance between cDC and pDC in the lung and its lymph nodes is crucial for the outcome of a pulmonary infection. Increased pDC numbers induced by Flt3L treatment have a protective impact on the nature of the overall immune environment.

Respiratory virus-induced TLR7 activation controls IL-17-associated increased mucus via IL-23 regulation.

The response to respiratory syncytial virus (RSV), negative strand ssRNA virus, depends upon the ability to recognize specific pathogen-associated targets. In the current study, the role of TLR7 that recognizes ssRNA was examined. Using TLR7−/− mice, we found that the response to RSV infection in the lung was more pathogenic as assessed by significant increases in inflammation and mucus production. Although there appeared to be no effect of TLR7 deficiency on type I IFN, the pathology was associated with an alteration in T cell responses with increases in mucogenic cytokines IL-4, IL-13, and IL-17. Examination of dendritic cells from TLR7−/− animals indicated a preferential activation of IL-23 (a Th17-promoting cytokine) and a decrease in IL-12 production. Neutralization of IL-17 in the TLR7−/− mice resulted in a significant decrease in the mucogenic response in the lungs of the RSV-infected mice. Thus, without TLR7-mediated responses, an altered immune environment ensued with a significant effect on airway epithelial cell remodeling and goblet cell hyper/metaplasia, leading to increased mucus production.

The post sepsis-induced expansion and enhanced function of regulatory T cells create an environment to potentiate tumor growth

One of the more insidious outcomes of patients who survive severe sepsis is profound immunosuppression. In this study, we addressed the hypothesis that post septic immune defects were due, in part, to the presence and/or expansion of regulatory T cells (Tregs). After recovery from severe sepsis, mice exhibited significantly higher numbers of Tregs, which exerted greater in vitro suppressive activity compared with controls. The expansion of Tregs was not limited to CD25(+) cells, because Foxp3 expression was also detected in CD25(-) cells from post septic mice. This latter group exhibited a significant increase of chromatin remodeling at the Foxp3 promoter, because a marked increase in acetylation at H3K9 was associated with an increase in Foxp3 transcription. Post septic splenic dendritic cells promoted Treg conversion in vitro. Using a solid tumor model to explore the function of Tregs in an in vivo setting, we found post septic mice showed an increase in tumor growth compared with sham-treated mice with a syngeneic tumor model. This observation could mechanistically be related to the ability of post septic Tregs to impair the antitumor response mediated by CD8(+) T cells. Together, these data show that the post septic immune system obstructs tumor immunosurveillance, in part, by augmented Treg expansion and function.

A novel inactivated intranasal respiratory syncytial virus vaccine promotes viral clearance without TH2 associated Vaccine-Enhanced disease

Background Respiratory syncytial virus (RSV) is a leading cause of bronchiolitis and pneumonia in young children worldwide, and no vaccine is currently available. Inactivated RSV vaccines tested in the 1960s led to vaccine-enhanced disease upon viral challenge, which has undermined RSV vaccine development. RSV infection is increasingly being recognized as an important pathogen in the elderly, as well as other individuals with compromised pulmonary immunity. A safe and effective inactivated RSV vaccine would be of tremendous therapeutic benefit to many of these populations. Principal Findings In these preclinical studies, a mouse model was utilized to assess the efficacy of a novel, nanoemulsion-adjuvanted, inactivated mucosal RSV vaccine. Our results demonstrate that NE-RSV immunization induced durable, RSV-specific humoral responses, both systemically and in the lungs. Vaccinated mice exhibited increased protection against subsequent live viral challenge, which was associated with an enhanced Th1/Th17 response. In these studies, NE-RSV vaccinated mice displayed no evidence of Th2 mediated immunopotentiation, as has been previously described for other inactivated RSV vaccines. Conclusions These studies indicate that nanoemulsion-based inactivated RSV vaccination can augment viral-specific immunity, decrease mucus production and increase viral clearance, without evidence of Th2 immune mediated pathology.

Generation of antifungal effector CD8+ T cells in the absence of CD4+ T cells during Cryptococcus neoformans infection

Immunity to the opportunistic fungus Cryptococcus neoformans is dependent on cell-mediated immunity. Individuals with defects in cellular immunity, CD4+ T cells in particular, are susceptible to infection with this pathogen. In host defense against a number of pathogens, CD8+ T cell responses are dependent upon CD4+ T cell help. The goal of these studies was to determine whether CD4+ T cells are required for the generation of antifungal CD8+ T cell effectors during pulmonary C. neoformans infection. Using a murine intratracheal infection model, our results demonstrated that CD4+ T cells were not required for the expansion and trafficking of CD8+ T cells to the site of infection. CD4+ T cells were also not required for the generation of IFN-γ-producing CD8+ T cell effectors in the lungs. In CD4− mice, depletion of CD8+ T cells resulted in increased intracellular infection of pulmonary macrophages by C. neoformans, increasing the pulmonary burden of the infection. Neutralization of IFN-γ in CD4−CD8+ mice similarly increased macrophage infection by C. neoformans, thereby blocking the protection provided by CD8+ T cells. Altogether, these data support the hypothesis that effector CD8+ T cell function is independent of CD4+ T cells and that IFN-γ production from CD8+ T cells plays a role in controlling C. neoformans by limiting survival of C. neoformans within macrophages.

Autophagy-Mediated Dendritic Cell Activation Is Essential for Innate Cytokine Production and APC Function with Respiratory Syncytial Virus Responses

The regulation of innate immune responses during viral infection is a crucial step to promote antiviral reactions. Recent studies have drawn attention to a strong relationship of pathogen-associated molecular pattern recognition with autophagy for activation of APC function. Our initial observations indicated that autophagosomes formed in response to respiratory syncytial virus (RSV) infection of dendritic cells (DC). To further investigate whether RSV-induced DC activation and innate cytokine production were associated with autophagy, we used several methods to block autophagosome formation. Using 3-MA, small interfering RNA inhibition of LC3, or Beclin+/− mouse-derived DC, studies established a relationship between RSV-induced autophagy and enhanced type I IFN, TNF, IL-6, and IL-12p40 expression. Moreover, autophagosome formation induced by starvation also promoted innate cytokine expression in DC. The induction of starvation-induced autophagy in combination with RSV infection synergistically enhanced DC cytokine expression that was blocked by an autophagy inhibitor. The latter synergistic responses were differentially altered in DC from MyD88−/− and TRIF−/− mice, supporting the concept of autophagy-mediated TLR signaling. In addition, blockade of autophagy in RSV-infected DC inhibited the maturation of DC as assessed by MHC class II and costimulatory molecule expression. Subsequently, we demonstrated that inhibition of autophagy in DC used to stimulate primary OVA-induced and secondary RSV-infected responses significantly attenuated cytokine production by CD4+ T cells. Thus, these studies have outlined that autophagy in DC after RSV infection is a crucial mechanism for driving innate cytokine production, leading to altered acquired immune responses.

B Cell Antigen Presentation Promotes Th2 Responses and Immunopathology during Chronic Allergic Lung Disease

Background The role of B cells in allergic asthma remains undefined. One mechanism by which B cells clearly contribute to allergic disease is via the production of specific immunoglobulin, and especially IgE. Cognate interactions with specific T cells result in T cell help for B cells, resulting in differentiation and immunoglobulin secretion. Proximal to (and required for) T cell-dependent immunoglobulin production, however, is antigen presentation by B cells. While interaction with T cells clearly has implications for B cell function and differentiation, this study investigated the role that B cells have in shaping the T cell response during chronic allergic lung disease. Methodology/Principal Findings In these studies, we used a clinically relevant mouse model of chronic allergic lung disease to study the role of B cells and B cell antigen presentation in this disease. In these studies we present several novel findings: 1) Lung B cells from chronically allergen challenged mice up-regulated MHC II and costimulatory molecules CD40, CD80 and CD86. 2) Using in vitro studies, B cells from the lungs of allergen challenged mice could present antigen to T cells, as assessed by T cell proliferation and the preferential production of Th2 cytokines. 3) Following chronic allergen challenge, the levels of Th2 cytokines IL-4 and IL-5 in the lungs and airways were significantly attenuated in B cell −/− mice, relative to controls. 4) B cell driven Th2 responses and mucus hyper secretion in the lungs were dependent upon MHC II expression by B cells. Conclusions/Significance Collectively, these results provide evidence for antigen presentation as a novel mechanism by which B cells contribute to chronic allergic disease. These findings give new insight into the mechanisms by which B cells promote asthma and other chronic diseases.

CxCL10/CxCR3-mediated Responses Promote Immunity to Respiratory Syncytial Virus Infection by Augmenting Dendritic Cell and CD8+ T Cell Efficacy

The induction of inflammatory cytokines during respiratory viral infections contributes to both disease pathogenesis and resolution. The present studies investigated the role of the chemokine CXCL10 and its specific receptor, CXCR3, in the host response to pulmonary respiratory syncytial virus (RSV) infection. Antibody‐mediated neutralization of CXCL10 resulted in a significant increase in disease pathogenesis, including airway hyperresponsiveness (AHR), mucus gene expression, and impaired viral clearance. When the pulmonary cytokine levels were examined, only type I IFN and IL‐12p70 were significantly reduced. These latter observations were reflected in reduced dendritic cell (DC) numbers and DC maturation in the lungs of RSV‐infected mice treated with anti‐CXCL10. Neutralization of the only known receptor for CXCL10, CXCR3, resulted in similar increases in pathogenic responses. When bone marrow‐derived DC were incubated with CXCL10 and RSV, an up‐regulation of type I IFN was observed. In addition, T lymphocytes were also examined and a significant decrease in the number of RSV M2 peptide‐specific CD8+ T cells was identified. These findings highlight a previously unappreciated role for the CXCL10:CXCR3 signaling axis in RSV‐infected animals by recruiting virus‐specific T cells into the lung and promoting viral clearance.