Neutrophil-Specific Knockdown of ß2 Integrins Impairs Antifungal Effector Functions and Aggravates the Course of Invasive Pulmonal Aspergillosis.

ß2-integrins are heterodimeric surface receptors that are expressed specifically by leukocytes and consist of a variable α (CD11a-d) and a common ß-subunit (CD18). Functional impairment of CD18, which causes leukocyte adhesion deficiency type-1 results in an immunocompromised state characterized by severe infections, such as invasive pulmonary aspergillosis (IPA). The underlying immune defects have largely been attributed to an impaired migratory and phagocytic activity of polymorphonuclear granulocytes (PMN). However, the exact contribution of ß2-integrins for PMN functions in-vivo has not been elucidated yet, since the mouse models available so far display a constitutive CD18 knockout (CD18-/- or CD18hypo). To determine the PMN-specific role of ß2-integrins for innate effector functions and pathogen control, we generated a mouse line with a Ly6G-specific knockdown of the common ß-subunit (CD18Ly6G cKO). We characterized CD18Ly6G cKO mice in-vitro to confirm the PMN-specific knockdown of ß2-integrins. Next, we investigated the clinical course of IPA in A. fumigatus infected CD18Ly6G cKO mice with regard to the fungal burden, pulmonary inflammation and PMN response towards A. fumigatus. Our results revealed that the ß2-integrin knockdown was restricted to PMN and that CD18Ly6G cKO mice showed an aggravated course of IPA. In accordance, we observed a higher fungal burden and lower levels of proinflammatory innate cytokines, such as TNF-α, in lungs of IPA-infected CD18Ly6G cKO mice. Bronchoalveolar lavage revealed higher levels of CXCL1, a stronger PMN-infiltration, but concomitantly elevated apoptosis of PMN in lungs of CD18Ly6G cKO mice. Ex-vivo analysis further unveiled a strong impairment of PMN effector function, as reflected by an attenuated phagocytic activity, and a diminished generation of reactive oxygen species (ROS) and neutrophil-extracellular traps (NET) in CD18-deficient PMN. Overall, our study demonstrates that ß2-integrins are required specifically for PMN effector functions and contribute to the clearance of A. fumigatus by infiltrating PMN, and the establishment of an inflammatory microenvironment in infected lungs.

Neutrophil extracellular traps impair fungal clearance in a mouse model of invasive pulmonary aspergillosis.

Neutrophil extracellular traps (NETs) are formed by polymorphonuclear neutrophils (PMN) and contribute to the innate host defense by binding and killing bacterial and fungal pathogens. Because NET formation depends on histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), we used PAD4 gene deficient (Pad4-/-) mice in a mouse model of invasive pulmonary aspergillosis (IPA) to address the contribution of NETs to the innate host defense in vivo. After the induction (24 h) of IPA by i.t. infection with Aspergillus fumigatus conidia, Pad4-/- mice revealed lower fungal burden in the lungs, accompanied by less acute lung injury, TNFα and citH3 compared to wildtype controls. These findings suggest that release of NETs contributes to tissue damage and limits control of fungal outgrowth. Thus inhibition of NETosis might be a useful strategy to maintain neutrophil function and avoid lung damage in patients suffering from IPA, especially in those suffering from preexisting pulmonary disease.

Antifungal Drugs Influence Neutrophil Effector Functions.

There is a growing body of evidence for immunomodulatory side effects of antifungal agents on different immune cells, e.g., T cells. Therefore, the aim of our study was to clarify these interactions with regard to the effector functions of polymorphonuclear neutrophils (PMN). Human PMN were preincubated with fluconazole (FLC), voriconazole (VRC), posaconazole (POS), isavuconazole (ISA), caspofungin (CAS), micafungin (MFG), conventional amphotericin B (AMB), and liposomal amphotericin B (LAMB). PMN then were analyzed by flow cytometry for activation, degranulation, and phagocytosis and by dichlorofluorescein assay to detect reactive oxygen species (ROS). Additionally, interleukin-8 (IL-8) release was measured by enzyme-linked immunosorbent assay. POS led to enhanced activation, degranulation, and generation of ROS, whereas IL-8 release was reduced. In contrast, ISA-pretreated PMN showed decreased activation signaling, impaired degranulation, and lower generation of ROS. MFG caused enhanced expression of activation markers but impaired degranulation, phagocytosis, generation of ROS, and IL-8 release. CAS showed increased phagocytosis, whereas degranulation and generation of ROS were reduced. AMB led to activation of almost all effector functions besides impaired phagocytosis, whereas LAMB did not alter any effector functions. Independent from class, antifungal agents show variable influence on neutrophil effector functions in vitro Whether this is clinically relevant needs to be clarified.

Coincident airway exposure to low-potency allergen and cytomegalovirus sensitizes for allergic airway disease by viral activation of migratory dendritic cells.

Despite a broad cell-type tropism, cytomegalovirus (CMV) is an evidentially pulmonary pathogen. Predilection for the lungs is of medical relevance in immunocompromised recipients of hematopoietic cell transplantation, in whom interstitial CMV pneumonia is a frequent and, if left untreated, fatal clinical manifestation of human CMV infection. A conceivable contribution of CMV to airway diseases of other etiology is an issue that so far attracted little medical attention. As the route of primary CMV infection upon host-to-host transmission in early childhood involves airway mucosa, coincidence of CMV airway infection and exposure to airborne environmental antigens is almost unavoidable. For investigating possible consequences of such a coincidence, we established a mouse model of airway co-exposure to CMV and ovalbumin (OVA) representing a protein antigen of an inherently low allergenic potential. Accordingly, intratracheal OVA exposure alone failed to sensitize for allergic airway disease (AAD) upon OVA aerosol challenge. In contrast, airway infection at the time of OVA sensitization predisposed for AAD that was characterized by airway inflammation, IgE secretion, thickening of airway epithelia, and goblet cell hyperplasia. This AAD histopathology was associated with a T helper type 2 (Th2) transcription profile in the lungs, including IL-4, IL-5, IL-9, and IL-25, known inducers of Th2-driven AAD. These symptoms were all prevented by a pre-challenge depletion of CD4+ T cells, but not of CD8+ T cells. As to the underlying mechanism, murine CMV activated migratory CD11b+ as well as CD103+ conventional dendritic cells (cDCs), which have been associated with Th2 cytokine-driven AAD and with antigen cross-presentation, respectively. This resulted in an enhanced OVA uptake and recruitment of the OVA-laden cDCs selectively to the draining tracheal lymph nodes for antigen presentation. We thus propose that CMV, through activation of migratory cDCs in the airway mucosa, can enhance the allergenic potential of otherwise poorly allergenic environmental protein antigens.

CD11b Regulates Fungal Outgrowth but Not Neutrophil Recruitment in a Mouse Model of Invasive Pulmonary Aspergillosis.

ß2 integrin receptors consist of an alpha subunit (CD11a-CD11d) and CD18 as the common beta subunit, and are differentially expressed by leukocytes. ß2 integrins are required for cell-cell interaction, transendothelial migration, uptake of opsonized pathogens, and cell signaling processes. Functional loss of CD18-termed leukocyte-adhesion deficiency type 1 (LAD1)-results in an immunocompromised state characterized by frequent occurrence of severe infections. In immunosuppressed individuals Aspergillus fumigatus is a frequent cause of invasive pulmonary fungal infection, and often occurs in patients suffering from LAD1. Here, we asked for the importance of CD11b/CD18 also termed MAC-1 which is required for phagocytosis of opsonized A. fumigatus conidia by polymorphonuclear neutrophils (PMN) for control of pulmonary A. fumigatus infection. We show that CD11b-/- mice infected with A. fumigatus were unaffected in long term survival, similar to wild type (WT) mice. However, bronchoalveolar lavage (BAL) performed 1 day after infection revealed a higher lung infiltration of PMN in case of infected CD11b-/- mice than observed for WT mice. BAL derived from infected CD11b-/- mice also contained a higher amount of leukocyte-attracting CCL5 chemokine, but lower amounts of proinflammatory innate cytokines. In accordance, lung tissue of A. fumigatus infected CD11b-/- mice was characterized by lower cellular inflammation, and a higher fungal burden. In agreement, CD11b-/-PMN exerted lower phagocytic activity on serum-opsonized A. fumigatus conidia than WT PMN in vitro. Our study shows that MAC-1 is required for effective clearance of A. fumigatus by infiltrating PMN, and the establishment of an inflammatory microenvironment in infected lung. Enhanced infiltration of CD11b-/- PMN may serve to compensate impaired PMN function.