Immune protection is dependent on the gut microbiome in a lethal mouse gammaherpesviral infection

Abstract

Immunopathogenesis in systemic viral infections can induce a septic state with leaky capillary syndrome, disseminated coagulopathy, and high mortality with limited treatment options. Murine gammaherpesvirus-68 (MHV-68) intraperitoneal infection is a gammaherpesvirus model for producing severe vasculitis, colitis and lethal hemorrhagic pneumonia in interferon gamma receptor-deficient (IFNγR−/−) mice. In prior work, treatment with myxomavirus-derived Serp-1 or a derivative peptide S-7 (G305TTASSDTAITLIPR319) induced immune protection, reduced disease severity and improved survival after MHV-68 infection. Here, we investigate the gut bacterial microbiome in MHV-68 infection. Antibiotic suppression markedly accelerated MHV-68 pathology causing pulmonary consolidation and hemorrhage, increased mortality and specific modification of gut microbiota. Serp-1 and S-7 reduced pulmonary pathology and detectable MHV-68 with increased CD3 and CD8 cells. Treatment efficacy was lost after antibiotic treatments with associated specific changes in the gut bacterial microbiota. In summary, transkingdom host-virus-microbiome interactions in gammaherpesvirus infection influences gammaherpesviral infection severity and reduces immune modulating therapeutic efficacy.

Full text

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Immune protection is dependent
on the gut microbiome in a lethal
mouse gammaherpesviral infection
Jordan R. Yaron
1,2,13, Sriram Ambadapadi 1,2,13, Liqiang Zhang 1,2, Ramani N. Chavan3,
Scott A. Tibbetts4, Shahar Keinan 5, Arvind Varsani
3,6,7,8, Juan Maldonado
3,9,
Simona Kraberger2,3, Amanda M. Tafoya1,2, Whitney L. Bullard4, Jacquelyn Kilbourne1,2,3,
Alison Stern-Harbutte4, Rosa Krajmalnik-Brown3,10,11, Barbara H. Munk1, Erling O. Koppang12,
Efrem S. Lim
3* & Alexandra R. Lucas1,2,4*
Immunopathogenesis in systemic viral infections can induce a septic state with leaky capillary
syndrome, disseminated coagulopathy, and high mortality with limited treatment options. Murine
gammaherpesvirus-68 (MHV-68) intraperitoneal infection is a gammaherpesvirus model for producing
severe vasculitis, colitis and lethal hemorrhagic pneumonia in interferon gamma receptor-decient
(IFNγR−/−) mice. In prior work, treatment with myxomavirus-derived Serp-1 or a derivative peptide
S-7 (G305TTASSDTAITLIPR319) induced immune protection, reduced disease severity and improved
survival after MHV-68 infection. Here, we investigate the gut bacterial microbiome in MHV-68 infection.
Antibiotic suppression markedly accelerated MHV-68 pathology causing pulmonary consolidation and
hemorrhage, increased mortality and specic modication of gut microbiota. Serp-1 and S-7 reduced
pulmonary pathology and detectable MHV-68 with increased CD3 and CD8 cells. Treatment ecacy
was lost after antibiotic treatments with associated specic changes in the gut bacterial microbiota. In
summary, transkingdom host-virus-microbiome interactions in gammaherpesvirus infection inuences
gammaherpesviral infection severity and reduces immune modulating therapeutic ecacy.
Viral infections induce potent immune responses, an immunopathogenesis that can lead to severe complications
with sepsis or leaky capillary syndromes and very high mortality and limited eective treatments, a true unmet
clinical need. Sepsis has an associated risk of disseminated intravascular coagulation (DIC) with thrombosis,
hemorrhage and shock1–3. One such group of viruses with known severe complications are the gammaherpesvi-
ruses (GHV). e murine gammaherpesvirus-68 (MHV-68) is a widely used, well-controlled laboratory model
of GHV host-pathogen interaction with genetic similarity to the human viruses Epstein-Barr virus (EBV) and
Kaposi’s sarcoma-associated herpesvirus (KSHV)4.
Inammatory vasculitic syndromes (IVS) are a group of rare, heterogeneous and devastating inammatory
conditions of the body’s extensive system of blood vessels with increased morbidity, including sudden loss of
vision, aneurysm, aortic arch syndrome, stroke, and associated increases in mortality5–7. e etiology of many
systemic vasculitides is currently unknown, with proposed mechanisms ranging from induction by fungal spores
1Center for Personalized Diagnostics, The Biodesign Institute, Arizona State University, Tempe, Arizona, USA.
2Center for Immunotherapy, Vaccines and Virotherapy, The Biodesign Institute, Arizona State University, Tempe,
Arizona, USA. 3Center for Fundamental and Applied Microbiomics, The Biodesign Institute, Arizona State University,
Tempe, Arizona, USA. 4Department of Molecular Genetics & Microbiology, College of Medicine, University of Florida,
Gainesville, Florida, USA. 5Cloud Pharmaceuticals, Research Triangle Park (RTP), North Carolina, USA. 6School of
Life Sciences, Arizona State University, Tempe, Arizona, USA. 7Center of Evolution and Medicine Arizona State
University, Tempe, Arizona, USA. 8Structural Biology Research Unit, Department of Integrative Biomedical Sciences,
University of Cape Town, Rondebosch, Cape Town, South Africa. 9KED Genomics Core, Arizona State University,
Tempe, Arizona, USA. 10Swette Center for Environmental Biotechnology, The Biodesign Institute, Arizona State
University, Tempe, Arizona, USA. 11School of Sustainable Engineering and the Built Environment, Arizona State
University, Tempe, Arizona, USA. 12Department of Basic Sciences and Aquatic Medicine, Faculty of Veterinary
Medicine, Norwegian University of Life Sciences, Oslo, Norway. 13These authors contributed equally: Jordan R. Yaron
and Sriram Ambadapadi. *email: efrem.lim@asu.edu; arlucas5@asu.edu
OPEN
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to herpesviruses infections (e.g., zoster)7,8. A lethal IVS large vessel arteritis model which closely mimics human
Giant Cell Arteritis, Kawasaki’s disease and Takayasu’s arteritis can be induced by high dose intraperitoneal
MHV-68 infection of interferon gamma receptor knockout (IFNγR−/−) mice9. Many infected mice display exten-
sive pulmonary hemorrhage and consolidation, mimicking DIC in viral sepsis, which has a very high attendant
mortality10. A signicant number of mice also have marked colon dilatation reminiscent of toxic megacolon11, in
addition to aggressive inammatory cell invasion with hemorrhagic lung consolidation. Herpesvirus infections
have been associated with inammatory bowel diseases12–14 and gastrointestinal involvement in systemic vascu-
litides has been reported by other groups15,16. As other gastrointestinal inammatory conditions are associated
with, or driven by, microbiome changes, the gut microbiome may have a role in the pathogenesis of GHV and
specically MHV-68 infections.
We have previously reported that immune modulating proteins from myxomavirus (MYXV) provide a new
class of anti-inammatory treatments when delivered as recombinant protein or by ectopic expression aer
Adeno-associated virus (AAV) delivery17–20. MYXV is the causative agent of lethal myxomatosis in the European
rabbit (Oryctolagus cuniculus), expressing highly potent immune evasion proteins that act as virulence factors in
MYXV infections. MYXV is not a pathogen for mice or humans. When puried as isolated recombinant proteins,
these immune modulating biologics can modify disease progression in a wide range of inammatory diseases
in preclinical animals and man21. In prior work, the MYXV-derived serine protease inhibitor (serpin) Serp-1
and a Serp-1 reactive center loop (RCL) peptide S-7 (G305TTASSDTAITLIPR319) signicantly improved survival,
reducing pulmonary and aortic inammation as well as hemorrhagic lung consolidation aer MHV-68 infec-
tion10,22. Serp-1 targets the coagulation pathways, both thrombolytic and thrombotic, and has proven, highly
eective anti-inammatory functions23,24. Antibiotic treatment of IFNγR−/− mice prior to MHV-68 infection
abrogated the ecacy of S-7 treatment for lethal MHV-68 infections, leading to signicantly reduced survival.
erapeutically modied S-7 peptides (MPS7–8 and MPS7–9), designed based upon the serpin crystal structure,
maintained ecacy in this model with improved survival aer MHV-68 infection25. e MPS7 peptides have
predicted increased hydrogen bonds when compared to the native S-7 peptide25.
e pathophysiologic role of the gut bacterial microbiome in gammaherpesviral infections and on immune
modulating treatments has not previously been investigated. We report here a systematic examination of the role
of the gut bacterial microbiome in MHV-68 infection and on Serp-1 and Serp-1-derived S-7 peptide treatment
in MHV-68 infections.
Results
Serp-1 protein and S-7 peptide treatments improve survival after MHV-68 infection in
IFNγR−/− mice. MHV-68 infections establish latency with chronic infection in wildtype mice26, causing
only u-like symptoms. Conversely, intraperitoneal infection of mice with underlying immune deciency, as
for IFNγR−/− mice, results in a lethal large vessel vasculitis, colitis and hemorrhagic pneumonia with early mor-
tality9,27. Intranasal infection, an alternative model for MHV-68, is not a model for severe vasculitic syndromes.
Our group and others have used this intraperitoneal infection model of vasculitis to study the pathogenesis of
gammaherpesvirus host-pathogen interactions, as well as to investigate the potential for anti-inammatory treat-
ment of associated inammatory vasculitic syndromes10,11,22,25,28,29. Intraperitoneal infection of IFNγR−/− mice
with 12.5 × 106 pfu was performed with follow-up for 150 days to determine survival rates. MHV-68 infection
with administration of control saline treatment alone for 30 days was lethal, with a median survival of 41 days.
Serp-1 (100 ng/g) or S-7 (100 ng/g) intraperitoneal (IP) injections for 30 days improved survival to 60% at 150
days (p = 0.0022 and p = 0.0218, respectively) (Fig.1B). us, Serp-1 and S-7 protect IFNγR−/− mice aer lethal
MHV-68 infection10,22.
Loss of gut bacteria accelerates MHV-68 induced disease. We hypothesized that the gut bacterial
microbiome may play a role in the host-pathogen interactions of MHV-68 induced disease through transking-
dom interactions. We completed a systematic analysis of the eect of microbiome depletion on MHV-68 induced
disease progression (Fig.2A). Mice were maintained on medicated water containing a broad-spectrum cocktail
Figure 1. Serp-1 and Serp-1 RCL-derived peptide S-7 improve survival in lethal MHV-68 infection. (A)
Structure of Serp-1 with A-beta sheet (blue) and reactive center loop (RCL; red) highlighted. RCL-derived
peptide S-7 illustrated in green. (B) Kaplan-Meier curve depicting survival of IFNγR−/− mice infected with
MHV-68 and treated with saline (le; N = 12 and 5), Serp-1 (middle; N = 5 and 5), or S-7 (right; N = 5 and 5)
without or with antibiotics.
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of four antibiotics: bacitracin, gentamicin, streptomycin and ciprooxacin (Table1) in order to suppress gut bac-
terial populations. During this time the density of the gut bacteria (as measured by colony forming units [CFU]
per gram fecal pellet) was markedly reduced, validating a decrease in bacterial load (Fig.2B). Aer ten days, mice
were returned to normal drinking water for 24 hours, during which time the antibiotic treatment maintained sup-
pression of the microbiome (Fig.2B). Mice were subsequently infected by IP injection of MHV-68 and injected
with control saline daily for 30 days and survival assessed for up to 150 days(Fig. 1B).
Suppression of gut bacteria by antibiotic treatment markedly accelerated the course of MHV-68 infection.
Saline-treated mice exhibited a median survival of 41 days, while antibiotic-treated mice had a median survival of
19 days (p = 0.0411; Fig.1B). 16 S rRNA gene amplicon sequencing was subsequently performed on DNA extracts
from the large intestines of individual mice at 3 days post- MHV-68 infection in order to assess early changes in
the gut microbiota. We detected a distinct dysbiosis in mice pre-treated with antibiotics prior to MHV-68 infec-
tion versus mice with a stable bacterial microbiome prior to infection (Fig.2C). is dysbiosis was associated
with a non-statistically signicant trend towards increased diversity in amplicon sequence variant (ASV) richness
(Fig.2D; p = 0.329). Sub-population analysis revealed signicant changes in candidate ASVs in MHV-68 infected
mice that were associated with antibiotic treatment (Fig.2E, top panels; increased abundance aer antibiotics) or
with no antibiotic treatment (Fig.2E, bottom panels; decreased abundance aer antibiotics). ese results suggest
that the gut bacterial microbiome is a signicant determinant of MHV-68 induced disease progression.
Serp-1 and S-7 peptide treatment ecacy is dependent on the gut bacterial microbiota. We
next investigated the potential for dependence of Serp-1 and S-7 therapeutic ecacy on the gut bacterial microbi-
ome in MHV-68 infected IFNγR−/− mice. Mice were treated with medicated drinking water (Table1) for 10 days,
Figure 2. Antibiotic treatment accelerates MHV-68 lethality and alters the gut bacterial microbiome. (A)
Overview of experimental design. Mice are treated with a broad-spectrum antibiotic cocktail in their drinking
water for 10 days, placed on normal water for 1 day, infected with MHV-68 with or without treatments with
follow-up for survival. (B) Antibiotic-medicated water completely ablates bacterial microbiome contents during
the course of the 10-day pre-treatment, which recovers by three days aer removal of antibiotics. (C) 16 S
microbiome relative abundance (genus level) and (D) Bacterial alpha diversity (Shannon Index) is shown for
saline-treated mice with and without antibiotics. (E) Heatmap showing ASV abundance in mice treated with
saline in the presence or absence of antibiotic pre-treatment.
Antibiotic Dose Target class Mechanism of action
Bacitracin 1 g/L Gram-positive Cell wall synthesis inhibition88
Gentamicin 0.5 g/L Gram-negative and Gram-positive Ribosome inhibition89
Streptomycin 2 g/L Gram-negative and Gram-positive Ribosome inhibition90
Ciprooxacin 0.125 g/L Gram-negative and Gram-positive DNA replication inhibition91
Table 1. Antibiotic cocktail components.
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placed on normal water for 24 hours, then infected with MHV-68 and concurrently treated for 30 days with either
Serp-1 or S-7 by IP injection. Mice were followed for survival or 3-day follow-up (Fig.2A).
As previously reported, Serp-1 and S-7 peptide improved survival with 60% of mice surviving to 150 days
aer MHV-68 infection. In contrast, antibiotic-treated mice had accelerated disease, with a median survival of 19
(Serp-1; p = 0.0025; Fig.1B) and 18 days (S-7; p = 0.0339; Fig.1B). Serp-1 or S-7 ecacy aer antibiotic treatment
is not signicantly dierent from saline treatment aer antibiotics (Serp-1 + Abx vs Saline + Abx, p = 0.2012;
S-7 + Abx vs Saline + Abx, p = 0.9164). us, the ecacy of Serp-1 or S-7 treatment and protection in lethal
MHV-68 infections was dependent on the gut bacterial microbiome.
Microbiome analysis identifies candidate taxa associated with Serp-1 and S-7 treat-
ments. Based on the ndings that (1) antibiotic-treated mice accelerated and exacerbated MHV-68 disease,
reducing survival time and increasing mortality, and that (2) immune protection conferred by Serp-1 and S-7 was
highly sensitive to antibiotic treatment, we proled the gut bacterial microbiota of mice treated with Serp-1 or
S-7. 16S rRNA gene amplicon sequencing was performed on DNA extracts derived from the large intestines of
individual mice 3 days post-infection with MHV-68 and aer treatment with Serp-1 or S-7 (Fig.3). Serp-1 and
S-7 treated mice, in the absence of antibiotics, had a higher diversity of microbiota (median = 1.270, p = 0.0079;
median = 0.862, p = 0.1508respectively) than saline treated mice (median = 0.385) (Fig.3B). Principal coordi-
nate analyses indicate that although the gut bacterial microbiomes of Serp-1 and S-7 treated mice diered from
saline treated mice, there was variability within Serp-1 and S-7 treated mice (Fig.3C, Supplementary Fig.1B).
is nding was consistent with the phenotypic variability observed in vivo. Specically, there was partial protec-
tion by Serp-1 and S-7 treatment with 60% survival (Fig.1B).
We hypothesized that responsiveness to Serp-1 and S-7 treatment may be driven by the presence of “pro-
tective” ASVs (dierentially associated with Serp-1 and/or S-7 treatment) or the absence of “potentiator” ASVs
(dierentially associated with saline treatment). erefore, to identify potential microbe(s) responsible for the
microbiome-mediated protection by Serp-1 and S-7, we analyzed the gut bacterial microbiota for discriminant
ASVs that were dierentially represented in Serp-1 and/or S-7 treated mice compared to saline treated mice, in
the absence of antibiotics (Supplementary Fig.1C–E). Of the 15 ASVs identied as dierentially associated with
Serp-1/peptide treatments, ASV4, was consistently associated with both Serp-1 and S-7 treatment (Fig.3D). ASV4
was most closely-related to the sequence of Bacillus massiliogorillae30. Conversely, ASV1 (most closely-related to
Frondihabitans peucedani31) and ASV123 (most closely-related to Enterococcus saigonensis32) were identied from
8 ASV candidates as being associated with saline treatment when compared to both Serp-1 or S-7 treatments.
Based upon the fact that Serp-1 and S-7 protected mice in an antibiotic-dependent manner, we reasoned that
the relative abundance of the discriminant ASVs would be altered in mice that were pre-treated with antibiotics.
As predicted, the relative abundance of Serp-1- or S-7-associated ASVs was decreased in antibiotic pre-treated
mice, albeit to a more moderate extent in S-7 (Fig.3E,F, compare “No Abx” to “ + Abx”). ese results suggest
that the interplay between with bacterial microbiota like ASV4, that are associated with a protective phenotype,
and microbiota such as ASV1 and ASV123 that are associated with a patho-exacerbative phenotype, can inuence
the outcome of immune modulating treatments. In summary, responsiveness to immune modulating therapy in
MHV-68 induced disease is associated with specic alterations in the gut bacterial microbiota.
Antibiotic-dependent exacerbation of early lung pathology of MHV-68 induced disease. MHV-
68 persistent infection33 leads to severe hemorrhagic and consolidating pulmonary pathology34–36. Hence, we
investigated whether the pulmonary pathology induced by MHV-68 infection reected the antibiotic-dependent
exacerbation we observed in survival analysis. We performed quantitative morphometric analyses of lungs at an
early, 3-day follow-up aer infection. In the absence of antibiotics, considerable early pulmonary consolidation
and inammation was observed in MHV-68-infected lung tissue in IFNγR−/− mice treated with control saline
alone. is severe pulmonary pathology was considerably reduced by Serp-1 or S-7 peptide treatment (Fig.4A)10.
Early lung pathology was markedly worse aer antibiotic treatment in all conditions (saline, Serp-1 or S-7), with
most animals in the saline group also showing hemorrhage in aected regions.
Previous reports have demonstrated that thickening of the alveolar wall/septa and reduction of alveolar lumen
area are reliable indicators of pulmonary inammation in acute laboratory models37–39. In this study, quantitative
morphometry of the lungs revealed a signicant reduction in alveolar wall thickness in MHV-68-infected mice
treated with Serp-1 or S-7 versus saline-treated controls that was lost aer treatment with antibiotics (Fig.4B).
Without antibiotic pre-treatment and when compared to saline-treated controls, alveolar lumen area was signi-
cantly increased in mice treated with S-7 (p = 0.0067) with a trend toward an increase in mice treated with Serp-1.
Antibiotic treatment nullied any level of protection by treatment with S-7 (Fig.4C). No diagnostic pathology
nor signicant changes in inammatory cell inltrates on histological analysis was noted in the gut or aorta at
this early, 3-day follow-up time (not shown). ese results indicate that immune protection against pulmonary
inammation and early stage hemorrhage promoted by Serp-1 and S-7 treatment are microbiome dependent.
Serp-1 and S-7 promote an increased gut microbiome-dependent CD3+ occupancy in the lungs.
T-cell responses play a crucial role in limiting active infection and have been reported as central mediators for
managing chronic MHV-68 infection40–42. Hence, we examined early, acute phase CD3+ T-cell recruitment to the
lungs in MHV-68 infections. Antibiotic treatment signicantly reduced CD3+ cells in the lungs of saline-treated
mice (Fig.4E, p = 0.0155). Further, S-7 signicantly increased, and Serp-1 trended towards increasing, the detect-
able CD3+ cells in the lungs of infected mice (Fig.4D,E), indicating that the serpin-mediated protection against
MHV-68 induced disease is closely associated with increased pulmonary T-cell activity. Further staining indi-
cates no appreciable eect of Serp-1 and S-7 on pulmonary CD4+ cells (Fig.4F), while CD8+ staining showed
signicant increases with S-7 (p = 0.0312) and a strong trend towards increase with Serp-1 (p = 0.1734) treatment
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(Fig.4G). Remarkably, antibiotic treatment that depleted the gut microbiome led to a loss of this observed CD3
inltration-promoting eects and a loss of CD8 bias of both Serp-1 and S-7 (Fig.4E-G). Taken together, these
results suggest protection from MHV-68 induced disease via CD3 recruitment and surveillance in the lungs, with
a CD8 bias, enhanced by Serp-1- or S-7, proceeds via a microbiome-dependent mechanism.
Figure 3. 16 S microbiome analysis. (A) 16S microbiome relative abundance (genus level) is shown. (B)
Bacterial alpha diversity (Shannon Index) is shown. Statistical signicance was assessed by Mann-Whitney
test. (C) Principal coordinate analysis (PCoA) plot of weighted UniFrac distances. (D) Heatmap showing ASV
abundance in mice treated with saline, Serp-1 or S7, in the absence of antibiotics. ASV shown were identied by
dierential analyses to be associated with Serp-1 or S7 (top section), or saline (bottom section). (E) Heatmaps of
dierential ASV from (D) are compared in the absence or presence of antibiotics for Serp-1 treated mice (le)
or S-7 treated mice (right). (F) Relative abundance of ASV4 (identied as dierentially associated with Serp-1
and S-7 treatment), and ASV1 and 123 (identied as dierentially associated with saline) in individual mouse
microbiomes. ASV taxonomic classication of the most closely related bacterial taxa are shown. Saline + Abx,
N = 6; Saline No Abx, N = 6; Serp-1 + Abx, N = 6; Serp-1 No Abx, N = 5; S7 + Abx, N = 6; S7 No Abx, N = 5.
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Serp-1 and S-7 reduce pulmonary MHV-68 levels in a gut bacterial microbiome-dependent
manner. Because T-cells are crucial for control of MHV-68 infection, we hypothesized that the promotion
of CD3 T-cell inltration would lead to reduced viral levels in the lungs. Antibiotic treatment did not change the
amount of MHV-68 antigen staining in the lungs of mice treated with saline control alone (Fig.5A,B). However,
Serp-1 and S-7 signicantly reduced MHV-68 antigen levels in the lungs in mice without antibiotic treatment
(Fig.5B; Serp-1, p = 0.0009; S-7, p = 0.0003). ese decreases in detectable MHV68 were partially reversed aer
antibiotic pre-treatment in MHV-68-infected mice (Figs.5 and S3). us, Serp-1 and S-7 initiate a gut bacterial
microbiome-dependent reduction in MHV-68 levels in the lungs.
Discussion
Compelling evidence has established an important role for the gut microbiome in immune responsiveness43–45 as
well as the pathogenesis of various diseases, including those with primary involvement outside of the gut, such
as respiratory46, hepatic47, renal48, neurologic49, autoimmune50, rheumatoid51 and cardiovascular52–54 conditions.
Some research groups have demonstrated that targeted manipulation of the gut microbiome to correct dysbiosis,
as for fecal transplants, may aid in treatment of certain diseases. For example, a small, randomized clinical trial
(NCT02636647) found improved outcomes in recurrent hepatic encephalopathy patients treated by fecal micro-
biota transplant from a single, rationally selected donor enriched with Lachnospiraceae and Ruminococcaceae
(taxa determined to be depleted in hepatic encephalopathy patients)55. A second trial (NTR1776) found that
fecal microbiota transfer from lean donors to obese donors partially reversed symptoms of metabolic syn-
drome, including improvements in insulin resistance56. Aside from correcting dysbiosis, recent evidence sup-
ports the hypothesis that microbiome composition also plays a signicant role in determining the ecacy of
some treatments. It was recently found that low gut microbiome levels of Akkermansia muciniphila caused
non-responsiveness in patients receiving PD-1 blockade immunotherapy for epithelial tumors57–59, reinforcing
the importance of the microbiome in treatment ecacy, as highlighted by a critical earlier study that identied a
positive correlation between Bacteroides fragilis and anti-CTLA4 therapy ecacy for melanoma60. Alternatively,
some treatments may inadvertently alter the gut microbiome and predispose to increased pathogenic infec-
tions. For example, it was recently demonstrated that proton pump inhibitors signicantly reduced microbial
diversity with changes in more than 20% of bacterial taxa, leading to increases in Enterococcus, Streptococcus,
Staphylococcus and the potentially pathogenic Escherichia coli species61. us, an understanding of how the gut
microbiome inuences, and is inuenced by, new therapeutic treatments is crucial.
Trans-kingdom interactions may also play a crucial role in dictating disease pathogenesis in mammalian hosts.
For viruses which require enteric bacteria for replication, antibiotics may reduce viral load. Numerous studies
have demonstrated this viral-bacterial interaction in laboratory systems, such as murine and human norovirus
Figure 4. Microbiome-dependent Serp-1 and S-7 ecacy is associated with pulmonary inammation and
increased occupancy of CD3+ and CD8+ cells (A) Representative H&E sections of mouse lungs at 3 days
follow-up aer MHV-68 infection without antibiotic pre-treatment (top row) or aer antibiotic pre-treatment
(bottom row) and with saline, Serp-1 or S-7 treatment. Scale bar is 100 µm. (B) Alveolar wall thickness and (C)
Alveolar lumen area of mouse lungs at 3 days follow-up as in (A). (D) Representative CD3 IHC micrographs
of mouse lungs at 3 days follow-up aer MHV-68 infection without antibiotic pre-treatment (top row) or
aer antibiotic pre-treatment (bottom row) and with saline, Serp-1 or S-7 treatment. Scale bar is 20 µm. (E)
Quantication of CD3+, CD4+ and CD8+ cells per 40× eld from 3–6 elds per mouse. Statistics in panels
B, C and E performed by Two-Way ANOVA with Fisher’s LSD post-hoc analysis. *p < 0.05, **p < 0.01,
***p < 0.001; n.s. is not signicant. N = 3 for all conditions.
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infection62,63. For other viruses, however, loss of the bacterial microbiome may lead to worsening clinical disease.
A recent study by ackray et al. reports that oral antibiotic (ampicillin) treatment exacerbates disease severity in
avivirus infections by modulating avivirus-specic CD8 responses64, suggesting a microbiome, immune system
and virus interaction. ese disparate observations, which depend substantially on (a) the virus being studied, (b)
the host system and (c) the antibiotic regimen, highlight how the virome is still a poorly understood, and conse-
quently under-appreciated, component of health and disease65.
We recently reported a severe gastrointestinal pathology in a signicant fraction of IFNγR−/− mice intraperi-
toneally infected with MHV-6811, a model for systemic arteritis and hemorrhagic pneumonia9,10. In this same
model, we also previously reported therapeutic ecacy with the MYXV-derived serine protease inhibitor Serp-
110 as well as peptides derived from the Serp-1 reactive center loop22,25. On this basis, we undertook a systematic
analysis of the eect of Serp-1 and the RCL-derived Serp-1 peptide S-7 on MHV-68 induced disease.
A broad-spectrum antibiotic cocktail was used to suppress the gut microbiome prior to MHV-68 infection.
We found that suppression of the gut microbiota prior to MHV-68 infection accelerated disease, with increased
lethality and earlier death occurring at approximately twice the rate of untreated mice (Fig.2C). is acceleration
of MHV-68 disease was accompanied by a dramatic post-antibiotic dysbiosis and a suite of candidate ASVs with
increased or decreased relative abundance associated with worsened disease outcomes.
Furthermore, we demonstrate here that bacterial microbiome interactions were required for a protective
immune response induced by Serp-1 or S-710. Microbiome ablation completely abolished treatment ecacy and
improved survival (Fig.1B). Probing further, we found that Serp-1 and S-7 facilitated protection against lung
inammation and consolidation associated with an increase in CD3+ cell invasion (with a CD8+ bias) and a
reduction of MHV-68 staining in the lungs (Figs.4 and 5). is nding is in agreement with previous reports that
T-cell surveillance (eected by CD8+ cells) is critical for limiting acute GHV infection and that a progressive
loss of T-cells is associated with worsened GHV disease40–42. e loss of Serp-1-based treatment ecacy was
not expected to be due to direct interactions between the antibiotics and immune modulating treatments for a
number of reasons: (1) the antibiotics used in this study have a relatively short half-life (1–7 hours) and previ-
ous studies report negligible concentrations 24 hours aer dosing66–69 – a timeline we incorporated in this study
design; (2) we observed a rapid recovery of microbial burden within three days (Fig.2B); (3) the antibiotics in
our cocktail are not known to suppress immune responses on their own, and in some reports are observed to
enhance T-cell activity70; (4) it is not expected that the loss of activity is due to direct interactions with the anti-
biotics because in vitro inhibition of uPA activity by Serp-1 is unaected by the presence of the same antibiotic
cocktail (Fig.S2). A recent report by Yang et al. indicated that in the short-term, antibiotics can induce metabolite
changes which have systemic, suppressant eects on immune function in the peritoneal space by altering respira-
tory activity44. Yang et al., however, utilized an E. coli infection and the antibiotic, ciprooxacin, was given within
4 hours of infection and maintained during the course of the infection. Here, we utilize a gammaherpesvirus
infection with longer pathogenic kinetics and remove antibiotics 24 hours prior to infection, wherein any trace of
ciprooxacin would be undetectable66, and we are not aware of any other reports suggesting immune suppressant
eects of the other antibiotics in our cocktail. Taken together, these data provide the rst evidence that microbi-
ome interactions are essential for a protective immune response, induced here by Serp-1 and S-7 treatment. We
have previously found Serp-1 to function through the urokinase-type plasminogen activator receptor (uPAR) in
systems involving immune cell inltration (e.g., wound healing71, chemokine-induced ascites72). Levels of uPAR
have been found to increase substantially in incidences of infection and sepsis73. us, future work will investigate
the possibility that the microbiome is modulating levels of binding partners essential for Serp-1 and S-7 function
in MHV-68 infection.
We propose here that transkingdom interactions with the gut bacterial microbiome are required for host
innate immunity to mount a protective response against lethal gammaherpesviral disease (Fig.6). When
Figure 5. Serp-1 and S-7 reduce MHV-68 presence in a gut bacterial microbiome-dependent manner (A)
Representative IHC images of 100 × elds depicting MHV-68 antigen staining in the lungs of mice at 3 days
follow-up aer MHV-68 infection without antibiotic pre-treatment (top row) or aer antibiotic pre-treatment
(bottom row) and with saline, Serp-1 or S-7 treatment. (B) Quantication of percent of MHV-68 positively
stained cells per 100 × eld from 3 elds per mouse. N = 3 for all conditions.
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MHV-68 infection causes viral pulmonary inammation and sepsis, as induced in the MHV-68 infection model,
host innate immune responses mediated by gut bacterial microbiota interactions leads to the recruitment of
immune cells (e.g. T-cells) to the lungs. However, antibiotic ablation of the gut bacterial microbiota alters patho-
logic outcome by preventing eective immune response responses leading to acceleration of MHV-68 inamma-
tory pathogenesis. Immunomodulatory treatments (like Serp-1 and S-7) can induce a stronger innate immune
response (such as T-cell recruitment) through (other) bacterial microbiota interactions. us, outcomes of GHV
infection are determined by the interplay of host innate immunity and microbiota interactions.
Interestingly, the gut microbiota of all Serp-1 and S-7 treated mice did not clearly cluster separately from
saline treated mice. us, the dynamics of selected increased or decreased microbiome ASVs is associated with
worsened pathology or alternatively protective immune modulation. Given that not all mice were fully protected
by Serpin treatment, we speculate that the gut microbiota heterogeneity of Serp-1- or peptide-treated mice may
reect the true variability in immunological outcomes (i.e. 60% protection). We found specic ASVs that were
either increased in response to Serp-1/peptide treatment (e.g. ASV4), or decreased when compared to saline
treatment (e.g. ASV1 and ASV123).
Although the current study design could not assess the predictive value of these discriminant ASVs on indi-
vidual mice survival outcomes, future studies will need to be performed in order to stratify the role of dierent
microbiome sub-populations by specic (i.e., single) antibiotic treatment as well as dened microbial recon-
stitution (e.g., with the Altered Shaedler Flora community or similar)74. Taken together, these ndings suggest
that the responsiveness to immune modulations in MHV-68 disease involves a combination of interactions with
protective microbiota and patho-exacerbative microbiota. Further work will be needed to establish a direct role
for bacteria related to these select ASVs to prove cause-and-eect relationships and to dene a precise mechanism
of bacterial microbiome inuence on immune responses in GHV disease.
With this study, we report that the pathogenicity of MHV-68 infection and eective treatment with the
immune modulating serpin, Serp-1, and serpin peptide, S-7, is substantially accelerated by suppression of the
gut bacterial microbiome with antibiotics. MHV-68 is a model virus for studying GHV pathogenesis and severe
viral sepsis4,75, therefore this nding may have implications for the pathogenesis and treatments of other GHV,
such as KSHV and EBV, where adverse responses to antibiotics are clinically recognized76. We further report here
the modulation of gut microbiome composition by an immune modulating protein and peptide. In conclusion,
(1) microbiome changes may have signicant impact on therapeutic immune modulating proteins77,78 and pep-
tides79,80, and (2), antibiotic treatment may increase severity of GHV infections.
Methods
Ethics statement. All animal studies conform to local and national guidelines for animal care and exper-
imentation. Protocols were approved by the local Institutional Animal Care and Use Committee (IACUC) of
University of Florida (#201604234_01).
Figure 6. Proposed model (Le) In uninterrupted lethal MHV-68 infection of IFNγR−/− mice, the gut
microbiome and immune response interact to mount an ultimately insucient response of cells such as T-cells
to aected tissues (e.g., the lungs), leading to severe disease and death. Antibiotics suppresses the immune
stimulatory eects of the bacterial microbiome, reducing further the immune response and worsening
the disease. (Right) In immune modulator-mediated protection by treatments such as Serp-1 or S-7, the
interactions leading to a suciently mounted immune response are enhanced, promoting an increased T-cell
inltration to aected tissues, reducing disease pathology and leading to survival.
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Animals. IFNγR−/− mice (B6.129S7-Ifngr1tm1Agt/J) were purchased from the Jackson Laboratory (Bar Harbor,
ME, USA). Animals were housed in barrier conditions at the University of Florida Animal Care Services vivar-
ium and bred under specic pathogen-free conditions. Mice were weaned at 3 weeks, maintained on a 12-hour
light-dark cycle and were fed water and standard rodent chow ad libitum.
Antibiotic treatment. At 4 weeks of age, IFNγR−/− mouse cohorts were transferred from the ABSL1 colony
to a separate, ABSL2 colony. Gut microbiome suppression was achieved by replacing standard drinking water
with autoclaved reverse osmosis water (obtained from the same animal care facility) containing an antibiotic
cocktail (Table1) composed of Streptomycin (2 g/L), Gentamicin (0.5 g/L), Bacitracin (1 g/L) and Ciprooxacin
(0.125 g/L) for 10 days. One day (24 hours) prior to infection, medicated water was replaced with standard animal
care facility water, which was maintained for the remainder of the experiment.
MHV-68 infection. On day 11 (aer 10 days of antibiotics) mice (5 weeks old) were infected with MHV-68 at
a dose of 12.5 × 106 PFU in 0.1 mL DMEM by intraperitoneal (IP) injection as previously described22,25,27,29. Mice
were returned to the colony and monitored for signs of distress for the duration of the experiment. Mice were
either followed for 150 days to determine survival or euthanized at 3 days post-infection and organs harvested
into formalin for histology or RNAlater (ermo Scientic, USA) for microbiome analyses. Details on the num-
bers of MHV-68-infected mice in this study are detailed in Table2.
Gut bacterial microbiome sample processing. Large intestine samples preserved in RNAlater were
processed for total genomic DNA isolation using the ZymoBiomics® miniprep kit (Zymo Research) according
to manufacturer’s recommended procedure. Isolated DNA was quantied using a NanoDrop 2000C (ermo
Scientic) and stored at −80 °C. Samples were analyzed in the Arizona State University KED Genomics Core
for whole-sample 16 S rRNA gene amplicon sequencing. DNA library preparation for Illumina® MiSeq plat-
form was prepared according to the protocol from Earth Microbiome Project (http://www.earthmicrobiome.
org/emp-standard-protocols/16s). e 16 S primer set 515f-806r81 was used for 2 × 150 pair-ended sequencing.
16S rRNA gene amplicon sequencing and analysis. Illumina MiSeq sequencing reads (2 × 150 bp)
of the 16 S rRNA gene V4 region82 were analyzed with QIIME 2 (ver 2017.12)83 for 34 samples: Saline + Abx
(n = 6), Saline No Abx (n = 6), Serp-1 + Abx (n = 6), Serp-1 No Abx (n = 5), S7 + Abx (n = 6), S7 No Abx (n = 5).
Sequencing reads were processed with DADA2 to infer Amplicon Sequence Variants (ASVs) at a 97% identity
threshold using Greengenes database (version 13.8)84. To account for inter-sample depth variability, all samples
were rareed to 55,000 reads per sample (10 iterations) (Fig.S1A). One sample (12,107 reads; Saline No Abx
group) was omitted due to insucient reads and the remaining 33 samples were preserved for further analy-
sis. ASV richness, alpha diversity (Shannon’s Diversity Index) and beta diversity (UniFrac distance) were cal-
culated using QIIME 2. Statistical analyses (read depth, ASV richness and Shannon Index comparisons) were
performed with Mann-Whitney test. Bars are median ± standard error. P-value less than 0.05 was considered
signicant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s. is not signicant. PCoA was performed with
weighted unifrac distance in QIIME 2. To identify discriminating ASVs associated with the respective treatments,
LEfSe (Linear discriminant analysis Eect Size)85, DESeq. 2 (version 1.20.0)86 and likelihood ratio tests were
performed in R studio (Version 1.1.456) (Fig.S1C–E). Discriminating analyses were rst performed compared
Serp-1 (no Abx) to saline (no Abx) mice, S-7 (no Abx) to saline (no Abx) mice, and nally as a combined group of
Serpin-treated mice [Serp-1 (no Abx) and S-7 (no Abx)] to saline (no Abx mice). Discriminant ASVs identied in
these analyses were pooled and validated in heatmap and abundance analyses (Fig.4D–F).
Histopathology. Mice were euthanized at 3 days follow-up aer infection by carbon dioxide asphyxiation
followed by cervical dislocation. Organs were harvested and xed in 10% neutral buered formalin. Samples
were dehydrated through graded alcohol, paraffin-embedded, sectioned into 4–6 µm ribbons, captured on
Treatment Antibiotics (ABX) Follow-up # of mice
Saline
No 3 days 6
Yes 3 days 6
No 150 days 12
Yes 150 days 5
Serp-1
No 3 days 6
Yes 3 days 6
No 150 days 5
Yes 150 days 5
S-7
No 3 days 6
Yes 3 days 6
No 150 days 5
Yes 150 days 5
Total # Infected Mice 73
Table 2. MHV-68-infected mice in this study.
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charged glass and dried overnight at 37 °C prior to processing for histopathology. Slides were rehydrated, stained
with Gill’s hematoxylin No.3 and Eosin Y (H&E) according to standard procedure, dehydrated and mounted in
Cytoseal XYL (ermo Fisher Scientic, USA).
Quantitative morphometry. H&E-stained aorta, lung and colon sections were imaged with a 20 × /0.5NA
objective on an Olympus BX51 microscope equipped with an Olympus DP74 camera operated by cellSens
Dimensions v1.16 soware. Objective-calibrated measurements of alveolar septal thickness and lumen area were
collected using cellSens Dimensions. At least 50 measurements were collected and averaged for each mouse, and
at least three mice were examined for each group. Images were processed for visualization in gures using ImageJ/
FIJI v1.52i87.
Immunohistochemistry. FFPE blocks containing lung tissue were sectioned into 4–6 µm ribbons, cap-
tured on charged glass and dried overnight at 37 °C prior to processing. Slides were rehydrated and epitopes
retrieved by boiling in sodium citrated buer. Endogenous peroxidases were quenched with 3% hydrogen per-
oxide, slides were blocked in 5% bovine serum albumin in TBS/0.1% Tween 20 and sections were probed with
rabbit polyclonal antibody against CD3 (Abcam ab5690; 1:200), CD4 (Abcam ab183685; 1:1000) or CD8 (Abcam
ab209775; 1:2000). For MHV-68 detection, 1:500 rabbit anti-serum or pre-immune serum (as control) were used
for immunostaining (kind gi of Dr. H.W. Virgin III9). 1:500 goat anti-rabbit HRP was used for secondary stain-
ing (Jackson Immuno Research 111-035-144). Antigens were revealed with ImmPACT DAB (Vector Labs, USA)
and mounted with Cytoseal XYL. Sections were imaged with a 40 × /0.75NA objective.
Viral load determination by qPCR. Total DNA was isolated from each FFPE sample (4 × 5 µm thick
sections for each sample was used) using the QIAamp DNA FFPE Tissue Kit according to the manufacturer’s
instructions. Samples were quantied using a DS-11 series spectrophotometer/uorometer (Denovix, USA).
Quantitative PCR (qPCR) was undertaken to investigate relative viral load using the following reaction per sam-
ple: 10 µL SsoAdvanced Universal SYBR Green Supermix (Bio-rad Laboratories Inc., USA), 0.4 µl each of the
primers 65 F (5′-GTCAGGGCCCAGTCCGTA-3′) and 65 R (5′-TGGCCCTCTACCTTCTGTTGA-3′), 200 ng of
DNA and water up to 20 µL total volume. Reactions were run in triplicate with controls, and a standard curve
using pCR2.1 Topo plasmid (ermo Fisher Scientic, USA) containing the cloned target MHV region35. e
following cycling conditions used on a CFX96 (Bio-rad Laboratories, Inc., USA) instrument: 95 °C for 20 sec, 40
cycles (95 °C for 15 sec, 60 °C for 20 sec), followed by a melt curve analysis.
Statistical analysis. Survival and pathology statistics were analyzed with GraphPad Prism v8.0.1.
Kaplan-Meier survival statistics were calculated using Log-rank (Mantel-Cox) testing. For visualization, individ-
ual comparison curves are presented. Lung pathology statistics were compared using a Two-Way ANOVA with
a Fisher’s LSD or Tukey’s post-hoc test. Bars are mean ± standard error. P-value less than 0.05 was considered
signicant. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; n.s. is not signicant.
Data availability
Sequence data has been deposited to the NCBI Sequence Read Archive under BioProject accession number
PRJNA517927.
Received: 1 August 2019; Accepted: 22 January 2020;
Published: xx xx xxxx
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Acknowledgements
e authors gratefully acknowledge Dr. Grant McFadden and Dr. Christian Jobin for many helpful discussions.
The authors acknowledge resources and support from the KED Genomics Core, part of the Biosciences
Core Facilities at Arizona State University. This study was financially supported by grants from the NIH
(1R01AI100987-01A1 and 1RC1HL100202), American Heart Association (17GRNT33460327), University of
Florida Gatorade Fund (00115070) and start-up funds from the Biodesign Institute at Arizona State University
all to ARL.
Author contributions
J.R.Y., S.A., L.Z., and A.R.L. designed research; J.R.Y., L.Z., R.C., E.L., A.V., S.Kraberger., J.M., R.K-B., B.H.M.
E.O.K. and A.R.L. analyzed data. J.R.Y. S.A. L.Z. S.Kraberger., A.S-H., A.M.T. and J.K. performed research; S.A.T.,
S.Keinan. and W.B. contributed new reagents or analytic tools; J.R.Y. and A.R.L. wrote the paper. All authors
reviewed the paper.
Competing interests
ARL is a listed inventor on issued patents US20060122115A1 and US7419670B2 on the therapeutic uses of
Serp-1, and on patent application US20180319868A1 on the therapeutic uses of Serp-1-derived RCL peptides.
ARL and JRY are listed inventors on provisional patent application 62/792,201and PCT/US20/13398 on
microbiome eects in inammatory vasculitis. ere are no other competing interests.
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