Differential cellular immune responses against Orientia tsutsugamushi Karp and Gilliam strains following acute infection in mice

Scrub typhus is a leading cause of febrile illness in endemic countries due to infection with Orientia tsutsugamushi (Ot), a seriously understudied intracellular bacterium. Pulmonary involvement associated with vascular parasitism in patients is common and can develop into life threatening interstitial pneumonia. The diverse antigenicity of Ot genotypes and inter-strain differences in genome content are connected to varied virulence and clinical outcomes; however, detailed studies of strain-related pulmonary immune responses in human patients or small animal models of infection are lacking. In this study, we have used two clinically prevalent bacterial strains (Karp and Gilliam) to reveal cellular immune responses in inflamed lungs and potential biomarkers of disease severity. The results demonstrate that outbred CD-1 mice are highly susceptible to both Karp and Gilliam strains; however, C57BL/6 (B6) mice were susceptible to Karp, but resistant to Gilliam (with self-limiting infection), corresponding to their tissue bacterial burdens and lung pathological changes. Multicolor flow cytometric analyses of perfused B6 mouse lungs revealed robust and sustained influx and activation of innate immune cells (macrophages, neutrophils, and NK cells), followed by CD4+ and CD8+ T cells, during Karp infection, but such responses were greatly attenuated during Gilliam infection. The robust cellular responses in Karp-infected B6 mice positively correlated with significantly early and high levels of serum cytokine/chemokine protein levels (CXCL1, CCL2/3/5, and G-CSF), as well as pulmonary gene expression (Cxcl1/2, Ccl2/3/4, and Ifng). In vitro infection of B6 mouse-derived primary macrophages also revealed bacterial strain-dependent immune gene expression profiles. This study provided the lines of evidence that highlighted differential tissue cellular responses against Karp vs. Gilliam infection, offering a framework for future investigation of Ot strain-related mechanisms of disease pathogenesis vs. infection control.


Introduction
Orientia tsutsugamushi (Ot), a severely neglected, tropical bacterial pathogen is a leading cause of febrile illness in the Rickettsiales order.It is estimated that this infection results in 150,000 global deaths per year and 1 million cases annually, encompassing over 1 billion people at risk of infection [1][2][3][4].This trombiculid mite-transmitted bacterium can cause subclinical infection or non-specific symptoms, including headache, fever, myalgia, rash, and regional lymphadenopathy [5].Patients with delayed or inappropriate treatment can develop into severe cases that can lead to acute respiratory distress syndrome, acute encephalitis syndrome, and multiorgan failure [3,5,6].There are no approved vaccines currently available for scrub typhus, mainly due to the paucity of knowledge associated with bacterial pathogenesis and host immunity.Research efforts and resources to study human immune responses to Ot infection and tissue-specific changes during disease progression are very limited, generally confined to analyses of patient sera and peripheral blood cells [7][8][9][10].Since the lungs of infected patients and experimental animals carry the highest bacterial burdens and pathological changes [11][12][13][14], there is a great need to reveal pulmonary innate and cellular immune responses, in the context of exposure to different Ot strains that are known to be associated with diverse disease outcomes.
Within the Rickettsiaceae family, Ot has a relatively large genome at 1.9-2.5 Mbp, with an astounding number of repeat sequences and conjugative elements [15].This is in stark contrast to its closely related Rickettsia species that tend to have small (typically 1.1-1.3Mbp) and surprisingly stable genomes [16].This proclivity for repeating elements is not uncommon among other intracellular bacteria spp., such as Wolbachia or Ehrlichia, which possess mobile genetic elements and tandem intergenic repeats, respectively [17,18].The unique characteristics of Ot may account for its high diversity, and consequently antigenicity and virulence.Seven geographically diverse Ot genotype groups have been identified, each of them composed of multiple serotypes, based on their major type-specific antigens (TSA56) [15,19].Multiple strains of Ot contribute to the overall global burdens of scrub typhus patients [15,19].While the geographic distribution of strains is predominantly region specific, Karp and Gilliam strains are of particular interest, as they are the etiological agent of 65% and 26% of clinical cases in endemic countries, respectively.Also, they can cause severe scrub typhus and death, even among antibiotic-treated patients [20].Yet, the comparative study of host immune responses to these two strains is necessary to dissect the unique immune signature in scrub typhus.
Improved murine and non-human primate models of scrub typhus have been developed in recent years; some of these models are designed to mimic natural bacterial transmission, acute tissue injury, pathologic and immunologic features [11,12,[20][21][22][23][24].Several murine-based reports conducted more than 10 years ago are especially relevant to this study, due to their consideration of antigenically distinct Ot strains in the context of Ot inoculation doses and/or host susceptibility.For example, while Ot TA716 strain led to non-lethal infection in BALB/c and C3H/He mice, a lower dose of Karp caused lethal infection in BALB/c and C3H/He mice; in contrast, a similar dose of Gilliam led to lethal infection in C3H/He mice, but not in BALB/ c mice [25][26][27].Yet, the mechanisms underlying such diverse infection outcomes have never been investigated at the tissue, cellular, or molecular levels.Given that Karp, Gilliam, and TA716 strains are all pathogenic in humans, causing severe clinical outcomes in some patients [28][29][30], a better understanding of the pathogenic mechanisms underlying host susceptibility to clinically prevalent Ot strains is in urgent need, especially in the context of advanced immunologic approaches and assays.
In this study, we investigated host susceptibility and clinical outcomes following inoculation with the comparable doses of Karp and Gilliam in inbred B6 vs. outbred CD-1 mice.While CD-1 mice showed high susceptibility to both Ot strains, as we and others have reported [13,14], B6 mice were susceptible to Karp, but highly resistant to Gilliam (with no signs of disease).Focusing on the B6 models, we perfused lungs and performed multi-color flow cytometric analyses of tissue-infiltrating immune cell subsets and their activation status.We revealed robust and sustained innate immune responses in Karp-infected mice, which positively correlated with cytokine levels in serum samples and the pulmonary proinflammatory gene expression profiles.Our data reveal Ot strain-dependent differences for lung innate and cellular immune responses in the context of distinct clinical outcomes of scrub typhus.This study contributes to the understanding of the cellular and molecular basis underlying immune responses and pathology related to the Ot strains, offering a framework for future investigations into disease control strategies.

Mouse infection and organ collection
Female Swiss Webster CD-1 outbred mice were purchased from Envigo (East Millstone, NJ).Female B6 mice were purchased from Jackson Laboratory (Bar Harbor, ME).Mice were maintained under specific pathogen-free conditions in the same room for 9-10 days and infected at 8-12 weeks of age.Infections were performed in the Galveston National Laboratory ABSL3 facility at UTMB.All tissue processing and analytic procedures were performed in BSL3 or BSL2 laboratories, respectively.All infections were performed by using the same bacterial stocks of Ot Karp or Ot Gilliam strain prepared from L929 cells, as described in our previous studies [31,32].Two independent studies for comparison between B6 and CD-1 mice were performed.B6 mice were inoculated i.v. with 5.6-6.8 × 10 4 focus forming units (FFU) of Karp or Gilliam stocks (200 μl), or with PBS (negative controls).CD-1 mice were inoculated i.v. with 3.4 × 10 4 FFU of the same Karp or Gilliam stocks (200 μl), or with PBS.The challenge dose used for CD-1 mice was lower than that of B6 mice, due to their higher susceptibility documented in our previous report [14].Mice were monitored daily for body weight, signs of disease, and disease scores.The disease score (ranged from 0-5) was based on an approved animal sickness protocol [21,32].The criteria included mobility/lethargy, hunching, fur ruffling, bilateral conjunctivitis, and weight loss: 0-normal behavior; 1-active, some weight loss (<5%); 2-weight loss (6-10%), some ruffled fur (between shoulders); 3-weight loss (11-19%), pronounced ruffled fur, hunched posture, erythema, signs of reduced food/water taken; 4weight loss (20-25%), decreased activity, bilateral conjunctivitis, showing signs of incapable to reaching food/water; 5-non-responsive (or weight loss of greater than 25%) animal need to be humanely euthanized.After blood collection, lungs were perfused, and then lungs and spleen were collected at days 4, 8, 12, and 32.For each mouse, the same corresponding lung lobes were used for the below comparative studies.Each time point (including the mock group) consisted of five mice per group.Samples (5/group) were inactivated for immediate and subsequent analyses, with mock samples serving as the controls.

Bacterial load quantification
Animal tissues were collected and stored in RNALater (Qiagen) at 4˚C overnight for inactivation and then stored at -80˚C.DNA was extracted from tissue or cell culture using the DNeasy Blood & Tissue Kit (Qiagen), following the manufacturer's instructions.For tissues, less than or equal to 30 mg was used for each extraction.Bacterial burdens were quantified via qPCR and normalized to total nanogram (ng) of DNA per μL of sample, as established in our previous study [11,23].iTaq Universal Probes Supermix (Bio-Rad) was used for qPCR.A portion of the 47-kDa gene was amplified using the primer pair OtsuF630 (5 0 -AACTGATTTTATTCA AACTAATGCTGCT-3 0 ) and OtsuR747 (5 0 -TATGCCTGAGTAAGATACGTGAATGGAATT-3 0 ) (IDT, Coralville, IA).PCR products were detected with a specific probe OtsuPr665 (5 0 -6F AM-TGGGTAGCTTTGGTGGACCGATGTTTAATCT-TAMRA) (Applied Biosystems, Foster City, CA) [11].Data were expressed as the copy number of 47-kDa gene per ng of DNA.The copy number for the 47-kDa gene was determined by serial dilution of known concentrations of a control plasmid containing a single-copy insert of the gene.

Quantitative reverse transcriptase PCR (qRT-PCR)
Total RNA was extracted from B6 mouse lung tissues or bone marrow-derived macrophages (MF) by using the RNeasy mini kit (Qiagen) and treated with DNase, according to the manufacturer's protocol.Mock-infected lung or cell culture was used as a negative control.cDNA was synthesized via the iScript cDNA synthesis kit (Bio-Rad).Target gene abundance was measured by qRT-PCR using a Bio-Rad CFX96 real-time PCR apparatus.SYBR Green Master mix (Bio-Rad) was used for all PCR reactions.The assay included: denaturing at 95˚C for 3 min followed with 40 cycles of: 10s at 95˚C and 30s at 60˚C.The 2 −ΔΔCT method was used to calculate relative abundance of mRNA expression.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as the housekeeping gene for all analyses.Primer sequences were obtained from the Harvard Medical School Primer Bank, and their validation has been proved in our previous studies [11,31,32,34,35].All the gene names and primer sequences are listed in S1 Table.

Histology
Lung tissues were fixed in 10% neutral buffered formalin and embedded in paraffin.Tissue sections (5-μm thickness) were stained with hematoxylin and eosin and mounted on slides, as in our previous reports [11,23].Sections were imaged under an Olympus BX53 microscope, and at least five random fields for each section were captured.

Infection of mouse bone marrow-derived MF
Bone marrow cells were collected from the tibia and femur of B6 mice and treated with red blood cell lysis buffer (Sigma Aldrich).MFs were generated by incubating bone marrow cells at 37˚C with 40 ng/ml M-CSF (Biolegend, San Diego, CA) in complete RPMI 1640 medium (Gibco) [21].Cell medium was replenished at days 3, 6, and 9, respectively; cells were collected at day 10.MFs (5 × 10 5 ) were seeded into 24-well plates and allowed to adhere for 3 h prior to infection.Bacteria were added at a multiplicity of infection (MOI) of 10 in 100 μl to the wells for 1 h with periodic rotating.Then, 900 μl of fresh medium was added per well, and plates were incubated at 37˚C with 5% CO 2 .All bacteria stocks used in cell cultures were the same as the mouse model infections.Stocks were prepared using L929 cells, as described in our previous studies [14,31,36].

Statistical analyses
All in vivo data are representative of two independent experiments and are presented as mean ± SEM.The normality test was performed by using Shapiro-Wilk test.At all times, oneway ANOVA was used to test the differences in outcomes among independent groups, where appropriate.After a significant F-test for the ANOVA model, S ˇı ´da ´k's multiple comparison test was used for specified comparisons between two Ot strains for each time.Dunnett's multiple comparisons test was used for the comparison of each infected group to the mock group.Differences between survival curves were analyzed by using the Log-ranked (Mantel-Cox) test.GraphPad Prism software 10 was used for data analysis.

Karp infection consistently causes greater disease severity and bacterial burdens in both B6 and CD-1 mouse models
To assess Ot strain-related virulence, we infected B6 mice with the same doses of Karp and Gilliam (6.8 ×10 4 FFU, i.v.) and monitored the mice daily.To validate the infectious nature of the Ot Gilliam strain and to identify potential differential host susceptibility to these two Ot strains, we also i.v.infected outbred CD-1 Swiss Webster mice with Karp or Gilliam strain (3.4 ×10 4 FFU).This relatively low infectious dose was selected based on our recent publication, which has indicated higher susceptibility of CD-1 mice than B6 mice to Karp [14].We found that CD-1 mice with Karp infection also began to lose weight (  1F).These changes were consistent with observation of clinical signs of disease, showing that Karpinfected mice developed significantly greater morbidity than Gilliam-infected ones.Collectively, the side-by-side comparison of inbred and outbred mouse models not only validated the infectivity of our bacterial stocks, but more importantly, demonstrated the diversity of bacterial virulence and host susceptibility in our established murine models (Karp-infected CD-1 > Gilliam-infected CD-1 >> Karp-infected B6 >> Gilliam-infected B6).

Karp, but not Gilliam, infection causes severe pulmonary damage during acute stage of infection
Having confirmed the diversity of host susceptibility to Karp vs. Gilliam infection, we decided to focus on the B6 models in the below study, for detailed analyses of host immune responses to two Ot strains.As shown in Fig 2A, bacterial burdens in Karp-infected lung tissues peaked at day 4, maintained at relatively high levels at day 8, and dropped substantially by day 12, with little to no bacteria detected at day 32.In sharp contrast, bacterial burdens within Gilliaminfected lung tissues were relatively low (peaked at day 12).Karp-infected lung tissues possessed 928-and 221-fold higher bacterial burdens at days 4 and 8 than those of Gilliaminfected lungs (statistically significant, p < 0.0001), respectively.Very similar temporal patterns were observed for Karp-and Gilliam-infected spleen samples; Karp-infected spleens had 9.69-and 8.32-fold higher bacterial burdens than those of Gilliam-infected spleens at days 4 and 8 respectively (Fig 2B).Therefore, Karp infection resulted in higher bacterial burdens in the lung and spleen tissues as compared to Gilliam infection during these acute stages of infection.
Given that pulmonary disease manifestations such as acute respiratory distress are common in scrub typhus patients [37], we examined lung pathology during murine infection with two Ot strains.As shown in Fig 3A, mock samples have no obvious inflammation and damage.Gilliam-infected lung tissues displayed mild inflammation with limited immune cell infiltration at day 8; the inflammation had lessened by day 12, comparable to the mock levels (Fig 3B).In

Karp infection results in robust innate and adaptive pulmonary immune responses
Having confirmed the rapid dissemination and growth of Karp strain and severe interstitial pneumonia associated with this infection, we then examined cellular immune responses in the lungs.At days 4, 8 and 12, we perfused lungs with PBS, prepared lung-derived single-cell suspension, and stained cells for flow cytometric analyses (S1 Fig, gating strategy).We found that while Karp-and Gilliam-infected lungs showed similar kinetics of immune cell accumulation, up to day 12, Gilliam-infected lungs had approx.40% less cell numbers than Karp-infected lungs at day 12 (p < 0.0001, Fig 4A).Analysis of lung innate immune cell subsets and activation status revealed several unique patterns.While both groups of mice showed nearly identical kinetics of monocyte accumulation during days 4-12 (Fig 4B), Karp-infected lungs had noticeably higher numbers of MFs (CD11b + CD64 + ) and significantly higher numbers of M1-type MFs (CD80 + CD206 -) at day 12 than Gilliam-infected lungs (p < 0.0001, Fig 4C ).Karp-infected lungs also had earlier and significantly higher influx of neutrophils (PMN, CD11b + Ly6G + ), as well as CD63 + activated PMNs at days 4 and 12, than Gilliam-infected lungs (Fig 4D).In addition, Karp, but not Gilliam infection showed progressive and significantly higher accumulation of NK cells (CD3 -NK1.1 + ) and activated NK cells (CD3 -NK1.1 + CD44 + ) at days 8 and 12 (Fig 4E).
Both CD4 and CD8 T cells play an important role in Ot elimination [8,38,39].As shown in Fig 4F, Karp-infected lungs showed accumulation of CD4 + T cells (CD3 + CD4 + ) and activated CD4 + T cells (CD3 + CD4 + CD44 + CD62L -), which were significantly higher than Gilliaminfected lungs at day 12 (p < 0.001 and p < 0.0001).Likewise in Fig 4G, accumulation of CD8 + T cells (CD3 + CD8 + ) and activated CD8 + T cells (CD3 + CD8 + CD44 + CD62L -) occurred at day 12, which were significantly higher than Gilliam-infected lungs at day 12 (p < 0.01).Overall, Karp-infected lungs possessed robust and sustained innate immune cell responses, followed by strong CD4 + and CD8 + T cell responses, all of which consistently peaked at day 12 and were significantly higher than Gilliam-infected lungs.These early robust immune cell expansions in Karp-infected lungs, and to a lesser extent in Gilliam-infected lungs, may play a key role in the over-reactive inflammatory response and lead to more severe clinical manifestations and host mortality.

Karp infection results in high levels of serum cytokines and chemokines
To investigate the systemic inflammation by two strain infection, we measured cytokine and chemokine levels in sera by using a Bio-Plex assay.As shown in Fig 5A, Karp-infected mice showed statistically significantly elevated levels of CXCL1, CCL2, and G-CSF, especially at day 4; these three markers were also significantly increased in Gilliam-infected mice at day 8, but at lower levels than those of Karp groups (p < 0.01).By days 8 and 12, Karp-infected mice had significantly higher levels of CCL3 and CCL5 than those of Gilliam-infected mice (Fig 5B).Additional markers that were differentially and significantly expressed in one of our independent studies were shown in S2A Fig, suggesting a pattern of higher serum IFN-γ, IL-10, IL-17, and Eotaxin levels in Karp-infected mice around days 8-12.These serum cytokine/chemokine protein profiles were consistent with our findings in the pulmonary flow cytometry results, supporting differential cellular immune response profiles during acute infection with two Ot strains.Increased IL-5, IL-9, IL-13, and GM-CSF were only observed in some samples but did not reach any statistic differences (S2B Fig) .Other makers (e.g., IL-1α, IL-1β, IL-2, IL-3, IL-4, IL-6, and IL-12p70) were undetectable in our tested samples.

Karp infection induces higher lung proinflammatory gene expression as compared to Gilliam infection
To further define pulmonary inflammatory response, we examined the gene expression of key cytokine and chemokine genes known to contribute to severe scrub typhus [11].At day 4, Karp-infected lungs, but not Gilliam-infected lungs, showed significantly elevated expression of Cxcl1, Cxcl2, Ccl2, Ccl3, and Ccl4, as compared to uninfected controls (Fig 6).During days 4-12, the Ccl3 and Ccl4 levels were consistently higher in Karp-infected lungs than Gilliaminfected tissues, while both groups showed comparable kinetics and levels of Ccl5.Of note, Karp-infected lungs showed significantly higher levels of Ifng at days 8 and 12 than Gilliaminfected tissues.Together, these qRT-PCR findings corresponded to the above flow cytometry results and serum protein profiles, confirming Ot strain-related differential responses in cytokine and chemokine expression.

Unique MF proinflammatory gene profiles are induced by two Ot strains
Using the Karp strain, we previously reported that lung MF subsets and activation status are strongly associated with disease severity and host mortality [21,22], and that Karp-MF

Discussion
Scrub typhus is a seriously understudied tropical infectious disease, which has recently emerged outside of its traditional geographic areas [42,43].Understanding cellular immune responses in the major target organs and during acute stages of infection with different Ot strains has been difficult and requires urgent attention.For example, our understanding of the physiologic, antigenic, and virulence differences between Karp and Gilliam strains is very limited, partially due to the complexity and diversity of Ot genome landscapes and the lack of adequate genetic tools for bacterial manipulation [44,45].In this study, we conducted a detailed investigation in murine models with these two clinically important Ot strains and revealed pulmonary innate and cellular immune responses in the context of distinct clinical outcomes of disease in B6 mice.This study, in conjunction with our previous comparison of pulmonary cellular immune responses in Karp-infected CD-1 vs. B6 mice [14], helps us understand the potential mechanisms underlying severe scrub typhus vs. self-limited infection and provides several lines of new evidence.
Firstly, we showed murine host-dependent differences in susceptibility to Karp vs. Gilliam infection.While Karp-infected B6 mice showed progressive weight loss and higher disease scores, reaching 20% mortality rates, Gilliam-infected mice gained some weight and showed no signs of disease (Fig 1A -1C).Such distinct outcomes of infection were not due to the quality of Gilliam stocks, as an even lower inoculation dose of Gilliam was capable causing lethal outcomes in CD-1 mice (50% mortality rates, Fig 1F).These clinical outcomes were consistent with tissue bacterial burdens (Fig 2 ) and lung pathology (Fig 3).Corresponding to our recent report [14], we confirmed here that CD-1 mice were more susceptible to Karp strain than B6 mice, as mortality rates for CD-1 mice reached 100% at day 15 (even though their inoculation dose was lower than that of B6 mice).Therefore, our side-by-side comparison of inbred and outbred mouse models clearly indicated a dose-dependent host susceptibility to two Ot strains: Karp-infected CD-1 > Gilliam-infected CD-1 >> Karp-infected B6 >> Gilliam-infected B6 mice.It has been reported that patients infected with the Karp strain showed a significantly higher bacterial DNA load than those infected with Gilliam strain [46].Our comprehensive studies with four infection murine models are important, as they mimic a broad spectrum of scrub typhus outcomes.Our findings here have greatly expanded beyond previous reports for Ot strain-based studies, offering important murine models for future vaccine-and/or drugbased studies for the control of scrub typhus.
Our conclusion of host-dependent susceptibility is consistent with other murine models for comparison between Karp and Gilliam, which overwhelmingly show Karp is more virulent in the animals, regardless of the routes of infection or mouse strain/stock used [13,26,27,43,[47][48][49][50].For non-human primate models, only a few comparison studies between bacterial strains have been conducted.For example, a recent study with rhesus macaques found that intradermal inoculated Gilliam can cause bigger skin lesions in comparison to Karp inoculation [20]; however, the immunologic or bacteriologic mechanisms underlying such differences have not been explored.Our study is the first to conduct a comparative analysis of lung immunology in B6 mice infected with virulent and less-virulent strains of Ot.The development and refinement of murine scrub typhus models, such as intradermal inoculation that can mimic the natural infection route, hold significant potential in this regard.
Secondly, we revealed Ot strain-related differences in tissue and cellular immune cell responses during Karp vs. Gilliam infection.Our use of perfused pulmonary tissues and multicolor flow cytometry of lung-derived immune cell subsets showed a higher influx and activation of innate immune cell subsets in Karp-over Gilliam-infected mice (Fig 4).Pulmonary immune cell comparison between bacterial strains represented a novel assessment of host immune response at the cellular level, revealing the kinetics and activation status of key cell subsets during acute stages of infection.We have several important findings related to innate and adaptive immunity in Ot infection.We found that Karp-infected lungs showed progressive influx/activation of all examined cell types (MF, PMN, NK, CD4 + and CD8 + T cells).Nearly all of them peaked at day 12 when lung bacterial burdens were apparently under control (reduced by approx.40-fold when compared between day 12 vs.day 8, Fig 2).Such findings support immune-mediated pathology in severe scrub typhus.
We also observed the different innate immune responses induced by two Ot strains.As early as day 4, Karp-infected, but not Gilliam-infected, lungs showed increased numbers of MF, M1-type MF, PMN, activated PMN, and activated NK cell populations, indicating differential host responses at the level of activating myeloid and innate immune cell subsets.Our findings from mouse models were relevant to some observations made from scrub typhus patients.For example, Paris and colleagues have suggested that indicators of PMN activation and neutrophil extracellular trap (NET) formation were considerably increased in severe scrub typhus patients, compared to those with less severe disease [51].
We previously reported that adaptive immunity plays a critical role during acute Ot infection [38].In this study, we found that as early as day 8, Karp-, but not Gilliam-, infected lungs showed increased numbers of activated CD4 + , total CD8 + , and activated CD8 + T cells.In contrast, Gilliam-infected lungs showed increased activated T cell subsets only at day 12, but to a much lesser magnitude than Karp-infected lungs.These flow cytometric studies have confirmed and greatly expanded the findings reported in our recent studies with Karp-infected mice [21].The activation of CD4 + T cells in infected mice is consistent with recent study of polyfunctional CD4 + T cells in the whole blood of scrub typhus patients [52].More importantly, our study clearly revealed differential kinetics of cellular immune responses in the lungs during acute stages of infection with two Ot strains.Along with NET formation, M1-type MFs, activated NK, and CD8 + T cells can exacerbate tissue damage.The involvement of a proinflammatory state in severe scrub typhus has been noted in other mouse studies [11,14,21,23,41], as well as in scrub typhus patients [7,[53][54][55].We have reported that the stronger innate response in the Karp-infected lungs also promotes excessive T cell activation and responses, which in turn contribute to bacterial control, but also amplify immunopathogenesis [32].It will be important for future studies to distinguish the monocyte/MF functionality between different strain infections to define immune signatures and biomarker relevant to bacterial control and disease outcomes.
Thirdly, we showed differential expression of biomarkers in serum and lung samples during Karp vs. Gilliam infection.We provided evidence that early and high levels of serum CXCL1, CCL2, and G-CSF proteins were hallmarks for Karp-infected mice, which were positively correlated to lung transcript levels of Cxcl1, Cxcl2, and Ccl2 at day 4.These chemokines/cytokines might contribute to early and extended recruitment and/or activation of MFs and PMNs seen in the lungs (Fig 4).The importance of some of these proinflammatory factors, including CCL2 and CXCL1, have been established previously and are components of a Mincle-dependent activation response [41].CCL2 and its receptor, CCR2, have also been suggested to play a role in the influx and activation of monocytes into the lungs, increasing bacterial replication and advancement of interstitial pulmonary inflammation [56].These levels of host response factors were either markedly mitigated, or showed different kinetics during Gilliam infection, correlating with the greatly reduced bacterial burdens and mild lung pathology.In addition, Karp infection induced higher IFN-γ, Eotaxin, IL-17, and IL-10 at days 8 or 12, indicating the stronger immune responses by Karp at late stages of infection.Ot infection can also slightly increase serum IL-5, IL-9, IL-13, and GM-CSF levels; however, their roles in scrub typhus remain unclear.Future studies will be needed to investigate the contributions of specific immune cell subsets and their involvement in the distinct clinical outcomes.Identifying key signatures associated with these immune cell subsets, such as in the study by Luce-Fedrow et al. [50], could be useful for parsing strain differences, as well as a future clinical tool to differentiate signatures associated with protective or lethal outcomes [50].
Finally, we revealed differential trends in MF's kinetic responses in vitro to help understand host susceptibility to Karp and Gilliam strains (Fig 7).We report here the early and significantly higher gene expression of innate responses, such as Il1b, Mx2, Mincle, and Fcgr1 in Karp-infected MFs than those in Gilliam-infected cells.This finding supports our previous report that sensing Karp bacteria via Mincle (a C-type-lectin receptor) and FcγR (Mincle signaling partner) can amplify TNFα production and type 1-skewed proinflammatory immune responses [41].It is therefore possible that divergent inflammatory signature between Karp and Gilliam infection might be related to Mincle and its downstream signaling [40].Higher levels of Tnf, Cxcl1 and Ccl2 in Karp-infected MF were consistent with our in vivo results (Fig 6 ), showing the increased myeloid cell infiltration and activation in the lungs at early infection.In contrast, Gilliam infection tended to induce higher levels of Ccl3 and Ccl4 at either 24 h or 72 h, indicating a unique regulation of inflammatory gene profiles.Although both Ot strains repressed Arg1, the high levels of Egr2 (an exclusive M2 MF marker [21,57]) in Gilliam-infected cells suggest a potential of this strain to induce M2-like gene expression, which is in comparison to the Karp strain [21].It will be of great interest to further examine whether different Ot strains can induce unique MF activation and polarization profiles via using multi-omics approaches.Such investigation will help define key immune determinants of bacterial clearance, host immune responses, and disease outcomes.
As illustrated in Fig 8 , our mouse model studies from two Ot strains and improved experimental approaches have greatly extended our recent reports for Karp infection in B6 and CD-1 mice, further supporting a notion of type 1-skewed, but type 2-repressed, inflammation in lethal/severe scrub typus [11,14,22,35,41,58,59].The infection by highly virulent Karp strain can lead to uncontrolled bacterial dissemination and replication in the lungs during the early stages.This excessive replication can trigger the production of proinflammatory chemokines (CXLC1/2, CCL2/3, etc.), resulting in the infiltration of innate immune cells (NK, MF, neutrophils) and subsequent influx of activated CD4 + and CD8 + T cells.These robust proinflammatory responses induced collectively by Karp strain can cause severe damage to pulmonary tissues and result in edema, ultimately leading to the animal's death during later stages of the infection.In contrast, the less-virulent Gilliam strain can be effectively eliminated at early stages, presumably due to quick and well-regulated activation of innate and adaptive immune cells, leading to limited tissue damage and a subclinical outcome.
At present, the mechanism by which Ot bacteria are eliminated efficiently at early stages of infection is still unknown.In a recent report for Rickettsia parkeri, another closely related obligate intracellular pathogen, it is found that infection control is mediated by both antiviral-like and antibacterial responses, involving IFN-I and IFNγ, respectively [60].Knowing that IFNγ plays a prominent role in Ot control and pathogenesis [61], it will be important to further investigate whether early IFN signaling contributes to the diversity of bacterial clearance in Karp vs. Gilliam infection.Furthermore, it is unclear as to whether different Ot strains replicate differently in endothelial cells in vivo.Several reports including Mika-Gospodorz et al., compared Ot strains in the context of host responses, provide a powerful overview of hostpathogen interaction [62].Further studies, including the potential use of genetic and molecular tools, will help dissect the underlying mechanism of Ot diverse virulence in vitro, as well as in inbred vs, outbred mouse models.
In summary, we have shown that Ot Karp-infected mice produce a higher degree of disease severity, mortality, bacterial burden, and pathologic lesions than that in Gilliam-infected mice.Our findings of strong and uncontrolled activation of cellular immune responses in the lungs, despite of seemingly controlled tissue bacterial burdens at late stages of Karp infection, highlight the contributions of Ot strain-dependent, immunopathogenesis in acute tissue injury in scrub typhus.We have provided new evidence for key signaling molecules that are involved in mounting host innate responses and highlighted new research questions and directions.This study provides a framework where strain-specific differences can be analyzed and used for future mechanistic and therapeutic studies.
All p-values were adjusted by multiple comparisons.All tests were two-sided, with a significance level of 0.05.Statistically significant values are denoted as asterixis with * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001, respectively.Only statistically significant comparisons are shown in the corresponding figures.All raw data are shown in S2 Table.
Karp-infected mice began to lose body weight (Fig 1A) and show disease symptoms at day 8 (Fig 1B), and 10% of them succumbing to infection by day 12 (Fig 1C), while the remaining mice stopped weight loss at day 13, indicating a sublethal outcome.In sharp contrast, mice infected with Gilliam did not exhibit any weight loss, disease scores or lethality (Fig 1A-1C), indicating a highly resistant phenotype.
Fig 1D) and show disease symptoms at day 8 (Fig 1E) and were moribund by day 15 (Fig 1F).CD-1 mice with Gilliam infection began to lose weight at day 10 and reached 50% mortality by day 15, with the remaining mice recovering gradually (Fig 1D-

Fig 2 .Fig 3 .
Fig 2. Differential bacterial burdens in Ot Karp-vs.Gilliam-infected lungs and spleens.C57BL/6 mice were inoculated with Karp, Gilliam, or PBS (mock), as described in Fig 1. A) Lung and B) spleen tissues were respectively collected at days 4, 8, 12, and 32 (5/group); all samples were subjected to DNA extraction.qPCR was performed to measure copy numbers of the 47-kDa gene by using itaq universal probes supermix and Ot-specific primers/ probe.Shown are representative results from two independent studies with similar trends.Data are presented as mean ± SEM.One-way ANOVA was used for statistical analysis.S ˇı ´da ´k's multiple comparisons test was used for multiple comparisons of two strains at each time.***, p < 0.001; ****, p < 0.0001.https://doi.org/10.1371/journal.pntd.0011445.g002

Fig 4 .
Fig 4. Strong and sustained cellular immune responses in Ot Karp-infected lungs.C57BL/6 mice were inoculated with Karp, Gilliam, or PBS (mock), as described in Fig 1.At days 0, 4, 8, and 12 post-infection (5/group), mouse lungs were perfused with a PBS solution.Lung tissues were digested, prepared for single-cell suspension, and stained for flow cytometric analyses.A) Total cell numbers of lungs, B) monocytes, C) total macrophages and M1 macrophages, D) PMN and activated PMN, E) NK and activated NK cells, F) total CD4 + and activated CD4 + T cells, and G) total CD8 + and activated CD8 + T cells.Data are presented as mean ± SEM.Shown are representative results from two independent studies with similar trends.One-way ANOVA was used for statistical analysis.S ˇı ´da ´k's multiple comparisons test was used for multiple comparisons between two strains at each time.**, p < 0.01; ***, p < 0.001; ****, p < 0.0001.https://doi.org/10.1371/journal.pntd.0011445.g004

Fig 8 .
Fig 8. Graphical illustration of differential innate and cellular responses in the lungs during acute infection with Ot Karp vs. Gilliam strains.Karp infection causes high tissue bacterial burdens and triggers robust proinflammatory immune responses in the lungs.Strong and uncontrolled production of MF and PMN chemotactic factors (CXCL1/2, CCL2/3) and other NK-and T cell-recruiting/stimulating factors, especially at late stages of infection, collectively lead to type 1-skewed cellular responses, acute tissue injury, and host mortality.In contrast, Gilliam dissemination to the lungs and replication in C57BL/6 mice are relatively slow and self-limited, due to mild and balanced cellular responses in the lungs.This graphical illustration is created with BioRender.com.https://doi.org/10.1371/journal.pntd.0011445.g008 The University of Texas Medical Branch (UTMB) complies with the USDA Animal Welfare Act (Public Law 89-544), the Health Research Extension Act of 1985 (Public Law 99-158), the Public Health Service Policy on Humane Care and Use of Laboratory Animals, and the NAS Guide for the Care and Use of Laboratory Animals (ISBN-13).UTMB is a registered Research Facility under the Animal Welfare Act.It complies with NIH policy and has current assurance with the Office of Laboratory Animal Welfare.All procedures were approved by the Institutional Biosafety Committee, in accordance with Guidelines for Biosafety in Microbiological and Biomedical Laboratories.Infections were performed following Institutional Animal Care and Use Committee approved protocols (2101001 and 1902006) at UTMB in Galveston, TX.