Kottom, Theodore

ABSTRACTWe explored differential polarization of macrophages during infection using a rat model ofPneumocystispneumonia. We observed enhanced pulmonary M1 macrophage polarization in immunosuppressed (IS) hosts, but an M2 predominant response in immunocompetent (IC) hosts followingPneumocystis cariniichallenge. Increased inflammation and inducible nitric oxide synthase (iNOS) levels characterized the M1 response. However, macrophage ability to produce nitric oxide was defective. In contrast, the lungs of IC animals revealed a prominent M2 gene signature, and these macrophages effectively elicited an oxidative burst associated with clearance ofPneumocystis. In addition, duringP. cariniiinfection the expression of Dectin-1, a critical receptor for recognition and clearance ofP. carinii, was upregulated in macrophages of IC animals but suppressed in IS animals. In the absence of an appropriate cytokine milieu for M2 differentiation,Pneumocystisinduced an M1 response bothin vitroandin vivo. The M1 response induced byP. cariniiwas plastic in nature and reversible with appropriate cytokine stimuli. Finally, we tested whether macrophage polarization can be modulatedin vivoand used to help manage the pathogenesis ofPneumocystispneumonia by adoptive transfer. Treatment with both M1 and M2 cells significantly improved survival ofP. carinii-infected IS hosts. However, M2 treatment provided the best outcomes with efficient clearance ofP. cariniiand reduced inflammation.

ABSTRACT Pneumocystis pneumonia (PcP) is a significant cause of morbidity and mortality in immunocompromised patients. In humans, PcP is caused by the opportunistic fungal species Pneumocystis jirovecii . Progress in Pneumocystis research has been hampered by a lack of viable in vitro culture methods, which limits laboratory access to human-derived organisms for drug testing. Consequently, most basic drug discovery research for P. jirovecii is performed using related surrogate organisms such as Pneumocystis carinii , which is derived from immunosuppressed rodents. While these studies provide useful insights, important questions arise about interspecies variations and the relative utility of identified anti- Pneumocystis agents against human P. jirovecii . Our recent work has identified the histone acetyltransferase (HAT) Rtt109 in P. carinii (i.e., PcRtt109) as a potential therapeutic target for PcP, since Rtt109 HATs are widely conserved in fungi but are absent in humans. To further address the potential utility of this target in human disease, we now demonstrate the presence of a functional Rtt109 orthologue in the clinically relevant fungal pathogen P. jirovecii (i.e., PjRtt109). In a fashion similar to that of Pcrtt109 , Pjrtt109 restores H3K56 acetylation and genotoxic resistance in rtt109 -null yeast. Recombinant PjRtt109 is an active HAT in vitro , with activity comparable to that of PcRtt109 and yeast Rtt109. PjRtt109 HAT activity is also enhanced by the histone chaperone Asf1 in vitro . PjRtt109 and PcRtt109 showed similar low micromolar sensitivities to two reported small-molecule HAT inhibitors in vitro . Together, these results demonstrate that PjRtt109 is a functional Rtt109 HAT, and they support the development of anti- Pneumocystis agents directed at Rtt109-catalyzed histone acetylation as a novel therapeutic target for human PcP.