Targeting host tyrosine kinase receptor EphA2 signaling via small-molecule ALW-II-41-27 inhibits macrophage pro-inflammatory signaling responses to Pneumocystis carinii β-glucans

Abstract

Pneumocystis jirovecii, the fungus that causes Pneumocystis jirovecii pneumonia (PJP), is a leading cause of morbidity and mortality in immunocompromised individuals. We have previously shown that lung epithelial cells can bind Pneumocystis spp. β-glucans via the EphA2 receptor, resulting in activation and release of proinflammatory cytokines. Herein, we show that in vivo Pneumocystis spp. β-glucans activation of the inflammatory signaling cascade in macrophages can be pharmacodynamically inhibited with the EphA2 receptor small-molecule inhibitor ALW-II-41-27. In vitro, when ALW-II-41-27 is administrated via intraperitoneal to mice prior to the administration of highly proinflammatory Saccharomyces cerevisiae β-glucans in the lung, a significant reduction in TNF-alpha release was noted in the ALW-II-41-27 pre-treated group. Taken together, our data suggest that targeting host lung macrophage activation via EphA2 receptor-fungal β-glucans interactions with ALW-II-41-27 or other EphA2 receptor kinase targeting inhibitors might be an attractive and viable strategy to reduce detrimental lung inflammation associated with PJP.

Full text

Eukaryotic Cells | Short Form
Targeting host tyrosine kinase receptor EphA2 signaling
via small-molecule ALW-II-41-27 inhibits macrophage pro-
inammatory signaling responses to Pneumocystis cariniiβ-
glucans
Theodore J. Kottom,1,2 Eva M. Carmona,1,2 Andrew H. Limper1,2
AUTHOR AFFILIATIONS See aliation list on p. 8.
ABSTRACT Pneumocystis jirovecii, the fungus that causes Pneumocystis jirovecii
pneumonia (PJP), is a leading cause of morbidity and mortality in immunocompromised
individuals. We have previously shown that lung epithelial cells can bind Pneumocystis
spp. β-glucans via the EphA2 receptor, resulting in activation and release of proinam-
matory cytokines. Herein, we show that in vivo Pneumocystis spp. β-glucans activation
of the inammatory signaling cascade in macrophages can be pharmacodynamically
inhibited with the EphA2 receptor small-molecule inhibitor ALW-II-41-27. In vitro, when
ALW-II-41-27 is administrated via intraperitoneal to mice prior to the administration of
highly proinammatory Saccharomyces cerevisiae β-glucans in the lung, a signicant
reduction in TNF-alpha release was noted in the ALW-II-41-27 pre-treated group. Taken
together, our data suggest that targeting host lung macrophage activation via EphA2
receptor-fungal β-glucans interactions with ALW-II-41-27 or other EphA2 receptor kinase
targeting inhibitors might be an attractive and viable strategy to reduce detrimental lung
inammation associated with PJP.
KEYWORDS Pneumocystis, pneumonia, inammation, mycology
EphA2 receptor is a member of the receptor tyrosine kinase family. It is a trans
membrane receptor containing an extracellular region that binds activating ligands
(ephrins) and more recently discovered to bind fungal β-glucans (1–3), leading to
the activation of the intracellular tyrosine kinase domain (4). Others have shown the
specicity of EphA2 receptor for fungal β-glucans and activation (phosphorylation) via
binding of this receptor to zymosan-coated beads as well as Candida albicans, Aspergillus
fumigatus, and Rhizopus delemar fungal organisms and the absence of binding and
phosphorylation of the receptor by Staphylococcus aureus and Escherichia coli bacteria
(3). More recently, our lab has shown that puried recombinant EphA2 protein alone can
specically and signicantly bind Pneumocystis β-glucans, verifying that the protein is
a receptor for fungal β-glucans (1). Traditionally, the EphA2 receptor kinase pathway
has important roles in carcinogenesis, pathological angiogenesis, and inammation
in atherosclerosis (5–7). Expression of EphA2 receptor is high in both epithelial and
endothelial cells (4). More recently, this receptor pathway has also emerged as an
important regulatory pathway for host defense against microbial pathogens, including
bacterial, viral, and fungal pathogens (2, 8–11). For example, it has been demonstrated
previously that the EphA2 receptor is active in the binding and trapping of the hook
worm Nippostrongylus brasiliensis by bone marrow-derived macrophages, suggesting a
role for the EphA2 receptor in macrophage/microbial pathogenesis (12).
February 2024 Volume 68 Issue 2 10.1128/aac.00811-23 1
Editor Helen Boucher, Tufts University - New
England Medical Center, Boston, Massachusetts, USA
Address correspondence to Theodore J. Kottom,
kottom.theodore@mayo.edu.
The authors declare no conict of interest.
See the funding table on p. 8.
Received 19 June 2023
Accepted 3 December 2023
Published 11 January 2024
Copyright © 2024 American Society for
Microbiology. All Rights Reserved.

ALW-II-41-27 is a small-molecule inhibitor that has been demonstrated to selectively
bind to the ATP-binding pocket of the EphA2 receptor kinase domain (13). Although the
compound has been shown to have EC50 values on a number of kinases in vitro at 10 uM
(including CSF1R, DDR1/2, Kit, Lck, and PDGFRα/β) (14), this and other recent studies
show EC50 eects of the inhibitor at 200 nM or less on EphA2 kinase activity, allowing
more directed and targeted therapeutic dosing (14–16). The inhibitor has been used
in the past to inhibit cancer cell growth in vitro and in vivo (17–20) and more recently
to inhibit uropathogenic bacteria adherence to bladder epithelial cells and to reduce
the oxidative stress and proinammatory host response in LPS(lipopolysaccharide)-trea
ted colonic cells (16). Therefore, we evaluated this inhibitor to determine whether it
might serve as an alternative agent to reduce proinammatory events resulting from
interactions with Pneumocystis β-glucans through the EphA2 receptor kinase signaling
pathway in macrophages in vitro (1), as well as the inhibitor’s ability to reduce yeast
β-glucan-driven proinammatory cytokine release in the lung. Measured and timed
therapeutic inhibition of EphA2 signaling may aid in mitigating harmful downstream
inammatory events that result during anti-Pneumocystis pneumonia (PCP) treatment.
We and others have shown that killing of fungal organisms as a result of this action
exposes highly inammatory β-glucan carbohydrate (21–29).
Cells [2 × 105 RAW macrophages (American Type Culture Collection)] for each
experimental condition were plated in ve wells of a 96-well microtiter plate and
incubated for 4 hours. After 4 hours, ALW-II-41-27 purchased from Sigma-Aldrich was
pre-incubated with the RAW cells for 60 minutes. Next, 100 ug/mL of Pneumocystis
carinii (Pc) β-glucans (28) was added to the wells, and the plates were centrifuged at
500 × g to synchronize the carbohydrate/macrophage interactions. Plates were then
placed at 37°C for 60 minutes. Next, cells were washed with 1× PBS, lysed, and protein
quantication determined. Total proteins in equal amounts were loaded and separated
by polyacrylamide gel electrophoresis. Finally, proteins were transferred to nylon for
Western blotting and incubated with antibodies to phospho-p38 or ERK1/2 as well as
total p38 and ERK1/2 (Cell Signaling Technology) to demonstrate equal loading control.
Protein phosphorylation kinetics were quantied with Image Studio Lite (LI-COR). All
experiments were repeated four to ve times. Activation of MAPK (mitogen-activated
protein kinase) is well documented in macrophage responses to Pneumocystis infection
(29–31). Herein, we demonstrate that the specic EphA2 receptor inhibitor ALW-II-41-27
can indeed signicantly inhibit Pc β-glucan-induced phosphorylation of both p38
(Fig. 1A) and ERK1/2 (Fig. 2) in a dose-dependent manner measured by Western blot
with similar ALW-II-41-27 concentrations previously published for cervical, endometrial,
nasopharyngeal, and colonic cells inhibitor studies (13, 15, 16, 32). Western blots for
both phospho-p38 (Fig. 1B) and phospho-ERK1/2 (Fig. 2B) were quantied by densi
tometry analysis against their respective total proteins. Next, we wanted to determine
whether ALW-II-41-27 inhibition would not only result in decreased MAPK phosphoryla
tion but also result in downstream reduced secretion of the proinammatory cytokine
TNF-alpha. To determine this, RAW macrophages were seeded as above. After 4 hours,
ALW-II-41-27 was incubated with the RAW cells for 60 minutes. Cell media were removed,
and 100 ug/mL of Pc β-glucans plus ALW-II-41-27 compound was applied to the cells,
centrifuged as described above, and incubated for 18 hours at 37°C. Supernatants were
then collected and assayed for TNF-alpha by ELISA (26). As shown in Fig. 3A, ALW-II-41-27
signicantly reduced TNF-alpha release in a dose-dependent fashion. To determine if
ALW-II-41-27 could signicantly alter the inammatory potential of macrophages already
undergoing activated proinammatory cytokine release via Pc β-glucan stimulation, we
also added ALW-II-41-27 post 60 minutes after Pc β-glucan stimulation. Similar to Fig. 3A,
we also noted signicant suppression of RAW cell TNF-alpha release in a dose response
fashion in these experiments (Fig. 3B). Next, to conrm these ndings in primary cells,
mouse lung alveolar macrophages were isolated as previously described (31). After
allowing the macrophages to bind for 2 hours, ALW-II-41-27 was incubated with the
macrophages for 60 minutes. As stated above, the supernatant was removed, and Pc
Short Form Antimicrobial Agents and Chemotherapy
February 2024 Volume 68 Issue 2 10.1128/aac.00811-23 2

β-glucans (100 ug/mL) plus ALW-II-41-27 compound was added to the cells, centrifuged
as described above, and incubated for 18 hours at 37°C . Supernatants were then
collected and assayed for TNF-alpha by ELISA as noted above. Similar to what was shown
in Fig. 3, ALW-II-41-27 addition to Pc β-glucan-induced primary mouse lung alveolar
FIG 1 (A) ALW-II-41-27 can signicantly inhibit Pc β-glucan-induced ERK1/2 phosphorylation in a dose-dependent manner. Representative blot of four separate
experiments. (B) The phospho ERK1/2 signals were quantied with Image Studio Lite software and normalized to total ERK1/2 levels. Initial analysis was rst
performed with ANOVA. If ANOVA indicated overall dierences, subsequent group analysis was then performed by a two-sample unpaired Student t test for
normally distributed variables. Error bars show SD from the mean. *P < 0.05, ns, non-signicant; ANOVA, analysis of variance.
FIG 2 (A) ALW-II-41-27 can signicantly inhibit Pc β-glucan-induced p38 phosphorylation. Representative blot of ve separate experiments. (B) The phospho p38
signals were quantied with Image Studio Lite software and normalized to total p38 levels. Similar to phospho ERK1/2 quantitation above, analysis of phospho
p38 quantication by ANOVA was rst performed. If ANOVA indicated overall dierences, subsequent group analysis was then performed by a two-sample
unpaired Student t test for normally distributed variables. Error bars show SD from the mean. ****P < 0.0001. ANOVA, analysis of variance.
Short Form Antimicrobial Agents and Chemotherapy
February 2024 Volume 68 Issue 2 10.1128/aac.00811-23 3

macrophages signicantly reduced TNF-alpha cytokine release from these cells (Fig. 4),
conrming the ability of the compound to inhibit proinammatory response in native
lung alveolar macrophages. To determine if ALW-II-41-27 could also aect TNF-alpha
release from RAW macrophages infected with live P. murina organisms, 2 × 105 cells were
plated in duplicate wells of a 96-well plate for 4 hours as above. Next, ALW-II-41-27 was
incubated with the RAW cells for 60 minutes. Finally, P. murina organisms in the presence
of ALW-II-41-27 were applied at a multiplicity of infection of 2:1 to the cell supernatant,
and the plates centrifuged at 500 × g to synchronize the fungal organism/RAW cell
interactions and incubated for 18 hours at 37°C. Supernatants were then collected
and tested for TNF-alpha release. Similar to what we reported above for Pc β-glucan
alone, TNF-alpha was signicantly induced in the presence of live fungal organisms. The
addition of 1,000 nM of ALW-II-41-27 to the media prior to the addition of the fungal
organisms signicantly suppressed TNF-alpha release from the cultured macrophages
(Fig. 5). These results suggest the exciting possibility of targeting the EphA2 tyrosine
kinase receptor pathway in those individuals with active PCP to reduce exuberant lung
inammation. On this note, as an initial proof-of-concept experiment to determine if
ALW-II-41-27 administered to mice could inhibit fungal β-glucan-driven proinammatory
cytokine response in the lung, we pre-treated mice with intraperitoneal (IP) injections of
either ALW-II-41-27 or the vehicle control 20 hours prior to the addition of Saccharomyces
cerevisiae β-glucans. After 20 hours, the yeast β-glucans were administered via intratra
cheally (IT). The following day (24 hours), the mice were sacriced, and total lung protein
lysates measured for TNF-alpha by ELISA. Remarkably, we noted that ALW-II-41-27 could
indeed signicantly reduce TNF-alpha protein levels in the lungs versus the vehicle
control group in yeast β-glucan-challenged mouse lungs (Fig. 6).
FIG 3 ALW-II-41-27 administered 60 minutes prior to (A) or 60 minutes after (B) Pc β-glucans signicantly dampens RAW 264.7 production of TNF-alpha in
vitro. Data are the ±SEM for at least four separate experiments. The TNF-alpha data analysis was initially rst performed with ANOVA. If ANOVA indicated overall
dierences, subsequent group analysis was then performed by a two-sample unpaired Student t test for normally distributed variables. Error bars show SD from
the mean. *P < 0.05, **P < 0.01, and ****P < 0.0001. ANOVA, analysis of variance.
Short Form Antimicrobial Agents and Chemotherapy
February 2024 Volume 68 Issue 2 10.1128/aac.00811-23 4

A number of reports have shown the importance of the EphA2 receptor pathway
in organism attachment and host immune recognition to microbial pathogens (12, 33–
35). Recently, we also have reported that EphA2 can bind Pneumocystis glucans and
is involved in lung epithelial cell proinammatory response to the organism’s cell wall
carbohydrate (1).
FIG 4 In primary mouse lung alveolar macrophages, ALW-II-41-27 signicantly dampens production of TNF-alpha in vitro in
the presence of Pc β-glucans. Data are the ±SEM for at least three separate experiments. Initial analysis was rst performed
with ANOVA. If ANOVA indicated overall dierences, subsequent group analysis was then performed by a two-sample
unpaired Student t test for normally distributed variables. Error bars show SD from the mean. *P < 0.05, and ****P < 0.0001.
ANOVA, analysis of variance.
Short Form Antimicrobial Agents and Chemotherapy
February 2024 Volume 68 Issue 2 10.1128/aac.00811-23 5

In vitro and in vivo data presented here suggest promising preliminary evidence
that therapeutically targeting the EphA2 receptor in PCP-infected individuals undergo
ing standard anti-PCP treatment may provide additional anti-inammatory relief as a
result of fungal killing and the release of highly proinammatory β-glucans during the
treatment of Pneumocystis pneumonia.
FIG 5 ALW-II-41-27 can signicantly inhibit P. murina-induced TNF-alpha secretion in mouse macro
phages. Data represent the mean ± SEM for at least three separate experiments. Initial analysis was rst
performed with ANOVA. If ANOVA indicated overall dierences, subsequent group analysis was then
performed by a two-sample unpaired Student t-test for normally distributed variables. Error bars depict
the SD from the mean. *P < 0.05 and **P < 0.01. ANOVA, analysis of variance.
Short Form Antimicrobial Agents and Chemotherapy
February 2024 Volume 68 Issue 2 10.1128/aac.00811-23 6

FIG 6 The eects of IP injection of ALW-II-41-27 on Saccharomyces cerevisiae β-glucan proinammatory response in the
lung. Twenty hours prior to administering 100 ug/mL of S. cerevisiae β-glucans via IT, mice were administered 0.1 mg/kg
ALW-II-41-27 or the vehicle (Methocel) control via IP. After 18 hours post drug or vehicle treatment, mice were administered
another 0.1 mg/kg of ALW-II-41-27 via IP or vehicle stated. After 2 hours, mice were administered 100 ug/mL of S. cerevisiae
β-glucans via IT administration. The following day, mice were sacriced, and total lung protein lysates (200 ug total) measured
for TNF-alpha by ELISA. Bar graph represents the results from 11 to 12 mice per group. If ANOVA indicated overall dierences,
subsequent group analysis was then performed by two-sample unpaired Student t test for normally distributed variables.
Error bars show SD from the mean. ****P < 0.0001. ANOVA, analysis of variance.
Short Form Antimicrobial Agents and Chemotherapy
February 2024 Volume 68 Issue 2 10.1128/aac.00811-23 7

AUTHOR AFFILIATIONS
1Departments of Medicine and Biochemistry, Mayo Clinic College of Medicine, Rochester,
Minnesota, USA
2Thoracic Diseases Research Unit, Mayo Clinic College of Medicine, Rochester, Minnesota,
USA
AUTHOR ORCIDs
Theodore J. Kottom http://orcid.org/0000-0003-0364-3311
Andrew H. Limper http://orcid.org/0000-0001-5671-6874
FUNDING
Funder Grant(s) Author(s)
HHS | National Institutes of Health (NIH) 1R21AI181542-01 Eva M. Carmona
AUTHOR CONTRIBUTIONS
Theodore J. Kottom, Conceptualization, Data curation, Formal analysis, Funding
acquisition, Investigation, Methodology, Writing – original draft | Eva M. Carmona, Formal
analysis, Writing – review and editing | Andrew H. Limper, Conceptualization, Formal
analysis, Funding acquisition, Supervision, Writing – original draft
REFERENCES
1. Kottom TJ, Schaefbauer K, Carmona EM, Limper AH. 2022. EphA2 is a
lung epithelial cell receptor for pneumocystis β-glucans. J Infect Dis
225:525–530. https://doi.org/10.1093/infdis/jiab384
2. Swidergall M, Solis NV, Millet N, Huang MY, Lin J, Phan QT, Lazarus MD,
Wang Z, Yeaman MR, Mitchell AP, Filler SG. 2021. Activation of EphA2-
EGFR signaling in oral epithelial cells by Candida albicans virulence
factors. PLoS Pathog 17:e1009221. https://doi.org/10.1371/journal.ppat.
1009221
3. Swidergall M, Solis NV, Lionakis MS, Filler SG. 2018. EphA2 is an epithelial
cell pattern recognition receptor for fungal β-glucans. Nat Microbiol
3:1074. https://doi.org/10.1038/s41564-018-0188-5
4. Zapata-Mercado E, Biener G, McKenzie DM, Wimley WC, Pasquale EB,
Raicu V, Hristova K. 2022. The ecacy of receptor tyrosine kinase EphA2
autophosphorylation increases with EphA2 oligomer size. J Biol Chem
298:102370. https://doi.org/10.1016/j.jbc.2022.102370
5. Wilson K, Shiuan E, Brantley-Sieders DM. 2021. Oncogenic functions and
therapeutic targeting of EphA2 in cancer. Oncogene 40:2483–2495.
https://doi.org/10.1038/s41388-021-01714-8
6. Pasquale EB. 2010. Eph receptors and ephrins in cancer: bidirectional
signalling and beyond. Nat Rev Cancer 10:165–180. https://doi.org/10.
1038/nrc2806
7. Funk SD, Orr AW. 2013. Ephs and ephrins resurface in inammation,
immunity, and atherosclerosis. Pharmacol Res 67:42–52. https://doi.org/
10.1016/j.phrs.2012.10.008
8. Swidergall M, Solis NV, Wang Z, Phan QT, Marshall ME, Lionakis MS,
Pearlman E, Filler SG. 2019. EphA2 is a neutrophil receptor for Candida
albicans that stimulates antifungal activity during oropharyngeal
infection. Cell Rep 28:423–433. https://doi.org/10.1016/j.celrep.2019.06.
020
9. Su C, Wu L, Chai Y, Qi J, Tan S, Gao GF, Song H, Yan J. 2020. Molecular
basis of EphA2 recognition by gHgL from gammaherpesviruses. Nat
Commun 11:5964. https://doi.org/10.1038/s41467-020-19617-9
10. Zhang J, Yuan J, Wang L, Zheng Z, Ran H, Liu F, Li F, Tang X, Zhang J, Ni
Q, Zou L, Huang Y, Feng S, Xia X, Wan Y. 2020. MiR-26a targets EphA2 to
resist intracellular Listeria monocytogenes in macrophages. Mol Immunol
128:69–78. https://doi.org/10.1016/j.molimm.2020.09.016
11. de Boer ECW, van Gils JM, van Gils MJ. 2020. Ephrin-Eph signaling usage
by a variety of viruses. Pharmacol Res 159:105038. https://doi.org/10.
1016/j.phrs.2020.105038
12. Bouchery T, Volpe B, Doolan R, Coakley G, Moyat M, Esser-von Bieren J,
Wickramasinghe LC, Hibbs ML, Sotillo J, Camberis M, Le Gros G, Khan N,
Williams D, Harris NL. 2022. β-glucan receptors on IL-4 activated
macrophages are required for hookworm larvae recognition and
trapping. Immunol Cell Biol 100:223–234. https://doi.org/10.1111/imcb.
12536
13. Xiang Y-P, Xiao T, Li Q-G, Lu S-S, Zhu W, Liu Y-Y, Qiu J-Y, Song Z-H, Huang
W, Yi H, Tang Y-Y, Xiao Z-Q. 2020. Y772 phosphorylation of EphA2 is
responsible for EphA2-dependent NPC nasopharyngeal carcinoma
growth by Shp2/Erk-1/2 signaling pathway. Cell Death Dis 11:709. https:/
/doi.org/10.1038/s41419-020-02831-0
14. Choi Y, Syeda F, Walker JR, Finerty PJ, Cuerrier D, Wojciechowski A, Liu Q ,
Dhe-Paganon S, Gray NS. 2009. Discovery and structural analysis of Eph
receptor tyrosine kinase inhibitors. Bioorg Med Chem Lett 19:4467–
4470. https://doi.org/10.1016/j.bmcl.2009.05.029
15. Li X, Li D, Ma R. 2022. ALW-II-41-27, an EphA2 inhibitor, inhibits
proliferation, migration and invasion of cervical cancer cells via
inhibition of the RhoA/ROCK pathway. Oncol Lett 23:129. https://doi.
org/10.3892/ol.2022.13249
16. Zeng L, Li K, Wei H, Hu J, Jiao L, Yu S, Xiong Y. 2018. A novel EphA2
inhibitor exerts benecial eects in PI-IBS in vivo and in vitro models via
Nrf2 and NF-κB signaling pathways. Front Pharmacol 9:272. https://doi.
org/10.3389/fphar.2018.00272
17. Martini G, Cardone C, Vitiello PP, Belli V, Napolitano S, Troiani T,
Ciardiello D, Della Corte CM, Morgillo F, Matrone N, Sforza V, Papaccio G,
Desiderio V, Paul MC, Moreno-Viedma V, Normanno N, Rachiglio AM,
Tirino V, Maiello E, Latiano TP, Rizzi D, Signoriello G, Sibilia M, Ciardiello
F, Martinelli E. 2019. EphA2 is a predictive biomarker of resistance and a
potential therapeutic target for improving antiepidermal growth factor
receptor therapy in colorectal cancer. Mol Cancer Ther 18:845–855.
https://doi.org/10.1158/1535-7163.MCT-18-0539
18. Ruan H, Li S, Bao L, Zhang X. 2020. Enhanced YB1/EphA2 axis signaling
promotes acquired resistance to sunitinib and metastatic potential in
renal cell carcinoma. Oncogene 39:6113–6128. https://doi.org/10.1038/
s41388-020-01409-6
19. Amato KR, Wang S, Tan L, Hastings AK, Song W , Lovly CM, Meador CB, Ye
F, Lu P, Balko JM, Colvin DC, Cates JM, Pao W, Gray NS, Chen J. 2016.
EphA2 blockade overcomes acquired resistance to EGFR kinase
inhibitors in lung cancer. Cancer Res 76:305–318. https://doi.org/10.
1158/0008-5472.CAN-15-0717
Short Form Antimicrobial Agents and Chemotherapy
February 2024 Volume 68 Issue 2 10.1128/aac.00811-23 8

20. Amato KR, Wang S, Hastings AK, Youngblood VM, Santapuram PR, Chen
H, Cates JM, Colvin DC, Ye F, Brantley-Sieders DM, Cook RS, Tan L, Gray
NS, Chen J. 2014. Genetic and pharmacologic inhibition of EPHA2
promotes apoptosis in NSCLC. J Clin Invest 124:2037–2049. https://doi.
org/10.1172/JCI72522
21. Kutty G, Davis AS, Ferreyra GA, Qiu J, Huang DW, Sassi M, Bishop L,
Handley G, Sherman B, Lempicki R, Kovacs JA. 2016. β-glucans are
masked but contribute to pulmonary inammation during Pneumocystis
pneumonia. J Infect Dis 214:782–791. https://doi.org/10.1093/infdis/
jiw249
22. Sassi M, Kutty G, Ferreyra GA, Bishop LR, Liu Y, Qiu J, Huang DW, Kovacs
JA. 2018. The major surface glycoprotein of Pneumocystis murina does
not activate dendritic cells. J Infect Dis 218:1631–1640. https://doi.org/
10.1093/infdis/jiy342
23. Evans HM, Bryant GL, Garvy BA. 2016. The life cycle stages of Pneumocys
tis murina have opposing eects on the immune response to this
opportunistic, fungal pathogen. Infect Immun 84:3195–3205. https://
doi.org/10.1128/IAI.00519-16
24. Evans HM, Garvy BA. 2018. The trophic life cycle stage of Pneumocystis
species induces protective adaptive responses without inammation-
mediated progression to pneumonia. Med Mycol 56:994–1005. https://
doi.org/10.1093/mmy/myx145
25. Evans HM, Simpson A, Shen S, Stromberg AJ, Pickett CL, Garvy BA,
Pirofski L. 2017. The trophic life cycle stage of the opportunistic fungal
pathogen Pneumocystis murina hinders the ability of dendritic cells to
stimulate CD4+ T cell responses. Infect Immun 85:e00396-17. https://doi.
org/10.1128/IAI.00396-17
26. Kottom TJ, Hebrink DM, Jenson PE, Gudmundsson G, Limper AH. 2015.
Evidence for proinammatory β-1,6 glucans in the Pneumocystis carinii
cell wall. Infect Immun 83:2816–2826. https://doi.org/10.1128/IAI.00196-
15
27. Linke MJ, Ashbaugh A, Collins MS, Lynch K, Cushion MT. 2013.
Characterization of a distinct host response prole to Pneumocystis
murina Asci during clearance of pneumocystis pneumonia. Infect
Immun 81:984–995. https://doi.org/10.1128/IAI.01181-12
28. Hahn PY, Evans SE, Kottom TJ, Standing JE, Pagano RE, Limper AH. 2003.
Pneumocystis carinii cell wall β-glucan induces release of macrophage
inammatory protein-2 from alveolar epithelial cells via a lactosylcera
mide-mediated mechanism. J Biol Chem 278:2043–2050. https://doi.
org/10.1074/jbc.M209715200
29. Wang J, Wright TW, Gigliotti F. 2011. Immune modulation as adjunctive
therapy for Pneumocystis pneumonia. Interdiscip Perspect Infect Dis
2011:918038. https://doi.org/10.1155/2011/918038
30. Tachado SD, Zhang J, Zhu J, Patel N, Cushion M, Koziel H. 2007.
Pneumocystis-mediated IL-8 release by macrophages requires
coexpression of mannose receptors and TLR2. J Leukoc Biol 81:205–211.
https://doi.org/10.1189/jlb.1005580
31. Kottom TJ, Nandakumar V, Hebrink DM, Carmona EM, Limper AH. 2020.
A critical role for CARD9 in pneumocystis pneumonia host defence. Cell
Microbiol 22:e13235. https://doi.org/10.1111/cmi.13235
32. Hudecek R, Kohlova B, Siskova I, Piskacek M, Knight A. 2021. Blocking of
EphA2 on endometrial tumor cells reduces susceptibility to Vδ1 gamma-
delta T-cell-mediated killing. Front Immunol 12:752646. https://doi.org/
10.3389/mmu.2021.752646
33. Chainarin S, Jaihan U, Tapaopong P, Kongngen P, Kunkeaw N, Cui L,
Sattabongkot J, Nguitragool W, Roobsoong W. 2022. Overexpression of
hepatocyte EphA2 enhances liver-stage infection by Plasmodium vivax.
Sci Rep 12:21542. https://doi.org/10.1038/s41598-022-25281-4
34. Shaikh MS, Islam F, Gargote PP, Gaikwad RR, Dhupe KC, Khan SL,
Siddiqui FA, Tapadiya GG, Ali SS, Dey A, Emran TB. 2022. Potential EphA2
receptor blockers involved in cerebral malaria from Taraxacum ocinale,
Tinospora cordifolia, Rosmarinus ocinalis and Ocimum basilicum: a
computational approach. Pathogens 11:1296. https://doi.org/10.3390/
pathogens11111296
35. Prakash PS, Kruse A, Vogel C, Schagdarsurengin U, Wagenlehner F. 2022.
Targeting host tyrosine kinase receptor EphA2 signaling aects
uropathogen infection in human bladder epithelial cells. Pathogens
11:1176. https://doi.org/10.3390/pathogens11101176
Short Form Antimicrobial Agents and Chemotherapy
February 2024 Volume 68 Issue 2 10.1128/aac.00811-23 9