Role of Innate Immune Receptors in Paradoxical Caspofungin ActivityIn Vivoin Preclinical Aspergillosis

Abstract

This study investigated the possible mechanisms underlying the paradoxical caspofungin activity in vivo in preclinical aspergillosis. We evaluated the activity of escalating doses of caspofungin in vivo in different preclinical models of invasive aspergillosis, including mice deficient for selected innate immune receptors. The therapeutic efficacy of caspofungin in experimental invasive aspergillosis was strictly dose dependent, being observed at doses of 0.1 and 1 mg/kg of body weight depending on the experimental models. Paradoxical increase in pulmonary fungal burden as well as inflammatory pathology was observed at the highest dose of caspofungin (5 mg/kg), occurred independently of the so-called Eagle effect and susceptibility to caspofungin in vitro, and was contingent upon the presence of TLR2, Dectin-1, and TLR9. Increased expression of Dectin-1 and TLR9 were observed upon exposure to caspofungin in vitro and in vivo. Together, these findings suggest that the net activity of caspofungin in vivo is orchestrated by the activation, directly or indirectly, of multiple innate immune receptors. Invasive mold infections are major causes of morbidity and mortality among severely immunocompromised individuals (24,34). Aspergillus fumigatus is currently the most important airborne fungal pathogen and causes different kinds of disease, ranging from allergies to life-threatening infections depending on the immune status of the host (35). The risk of invasive Aspergillus infection is particularly high for patients with persistent neutropenia, graft-versus-host disease (especially with concomitant steroid therapy), and certain types of hematopoietic transplantation (24,26,34).The echinocandins are the newest addition to the arsenal against fungal infections. They demonstrate similar in vitro and in vivo activity against Aspergillus spp., but only caspofungin (CAS) is approved for treatment of patients who are intolerant of or refractory to other therapies (27,38). CAS has been evaluated in the salvage and primary therapy of invasive aspergillosis, and combination regimens incorporating an echinocandin were promising in the treatment of aspergillosis (11). Echinocandins exhibit a unique, fungus-specific mechanism of action by targeting 1,3-␤-D-glucan synthase (14,17,19), thereby inhibiting the biosynthesis of 1,3-␤-D-glucan, an important component of the fungal cell wall. In addition to having a structural role, ␤-glucan, as a fungal pathogen-associated molecular pattern (PAMP), exhibits potent immunostimulatory properties mediated by pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs), which are expressed on host cells. Dectin-1 is the main PRR that recognizes ␤-glucans. Following binding, it induces the activation of phagocytic and proinflammatory responses (13).Studies with Candida albicans and Saccharomyces cerevisiae have revealed an echinocandin-susceptible genetic network that conceals ␤-glucan from the immune system, thus limiting an effective host respons...

Caspofungin Increases Fungal Chitin and Eosinophil and γδ T Cell–Dependent Pathology in Invasive Aspergillosis

“…Previous studies detailed the in vitro “paradoxical effect” of fungal growth in the presence of high levels of caspofungin (20, 54), and a recent study demonstrated increased chitin synthase activity and fungal stress response activation in response to caspofungin that permits A. fumigatus growth despite the presence of drug-mediated cell wall remodeling (55). These in vitro results are supported by in vivo evidence from Moretti et al that demonstrated decreased caspofungin efficacy in mice with IA (20, 40). Similar to our results, they reported mouse strain-dependent differences in fungal burden, with BALB/c mice most susceptible to infection at any dose of caspofungin, and C57BL/6 mice exhibiting a drug dose-dependent response.…”

“…In addition to reporting increased mouse fungal burden in IA after caspofungin therapy, Moretti et al also provided mechanistic data that demonstrated a requirement for TLR2, TLR9, and Dectin-1 (40). Interestingly, TLR2 and TLR9 expression were required for macrophage secretion of IL-17A and IL-10 in response to purified chitin particles (56, 57).…”

“…S1D–F). Interestingly, Moretti et al reported increased neutrophils in the lungs of high-dose caspofungin-treated animals with IA (40), and these cells are known to express high levels of dectin-1 (59). However, in contrast to the cyclophosphamide induced immune suppression used in that study, we depleted neutrophils prior to and during infection.…”

“…Neutropenic mice were infected with 5x10 6 condia of A. fumigatus isolates by involuntary aspiration. Caspofungin was prepared in sterile DPBS at 5mg/Kg (high dose) or 1mg/Kg (low dose) (40) and was injected i.p. on a daily basis until mice were harvested.…”

Hitting the Caspofungin Salvage Pathway of Human-Pathogenic Fungi with the Novel Lasso Peptide Humidimycin (MDN-0010)

“…However, after introduction of CAS in clinical trials, no decrease in the mortality rate of patients with invasive aspergillosis was reported (6). Apparently, there are at least two drawbacks: (i) the increasing resistance against CAS (7) and (ii) the so-called paradoxical effect leading to attenuated activity of echinocandin antifungals at high concentrations (8,9). However, echinocandins still show promising features as the basis for a combined antifungal therapy because they are poor substrates for cytochrome (CYP) P450 enzymes and P-glycoproteins (10).…”