Identification of Key TRIM Genes Involved in Response to Pseudomonas aeruginosa or Chlamydia spp. Infections in Human Cell Lines and in Mouse Organs

Bacterial infections represent an unsolved problem today since bacteria can evade antibiotics and suppress the host’s immune response. A family of TRIM proteins is known to play a role in antiviral defense. However, the data on the involvement of the corresponding genes in the antibacterial response are limited. Here, we used RT-qPCR to profile the transcript levels of TRIM genes, as well as interferons and inflammatory genes, in human cell lines (in vitro) and in mice (in vivo) after bacterial infections caused by Pseudomonas aeruginosa and Chlamydia spp. As a result, the genes were identified that are involved in the overall immune response and associated primarily with inflammation in human cells and in mouse organs when infected with both pathogens (TRIM7, 8, 14, 16, 17, 18, 19, 20, 21, 47, 68). TRIMs specific to the infection (TRIM59 for P. aeruginosa, TRIM67 for Chlamydia spp.) were revealed. Our findings can serve as a basis for further, more detailed studies on the mechanisms of the immune response to P. aeruginosa and Chlamydia spp. Studying the interaction between bacterial pathogens and the immune system contributes to the search for new ways to successfully fight bacterial infections.


Introduction
The duration and clinical outcome of a bacterial infection are determined not only by the characteristics of pathogenic bacteria, but also by their interaction with the innate immune system, which also makes a significant contribution.An important part of the immune system is the TRIM family, which includes about 80 proteins with a common structural N-terminal TRIpartite motif [1].TRIMs are known to be actively involved in the antiviral response of innate immunity [2], while their role in the antibacterial response has not yet been studied enough.In recent years, there has been evidence on the involvement of TRIMs in the functioning of the innate immune system during bacterial infection [3][4][5][6].Chen et al. (2018) showed that the expression of twenty TRIM genes was decreased in the peripheral blood cells of active tuberculosis patients infected with Mycobacterium tuberculosis in comparison with healthy individuals [3].TRIM27 and TRIM22 were demonstrated to inhibit the survival of M. tuberculosis [4,5], whereas the knockout of TRIM14 in macrophages restricted the reproduction of M. tuberculosis [7].TRIM21, TRIM56, and TRIM65 regulated the innate immune response during Salmonella enterica invasion [8,9].The deficiency of TRIM32 was shown to significantly reduce bacteremia and pro-inflammatory cytokine production after Streptococcus suis infection [10].Therefore, the available data demonstrate that TRIMs are able to both suppress bacterial infections or to serve as drivers of bacterial invasion.
The two types of pathogenic bacteria we have chosen for our study (Pseudomonas aeruginosa and Chlamydia spp.) are of particular interest, since the infections they cause are widespread in the world, especially among people with weakened immune systems [11,12].They differ in the way they interact with the cell and refer to extracellular (Pseudomonas aeruginosa) and obligate intracellular (Chlamydia spp.) pathogens.Both bacteria are able to induce and constitutively maintain an inflammatory response, as well as bypass the host's immune defense mechanisms.P. aeruginosa is an extremely problematic causative agent of respiratory and urinary diseases, with a whole arsenal of virulence factors aimed at suppressing the immune response.
We hypothesized that TRIMs could be actively involved in the immune system's response to these infections.In our work, we studied the expression profiles of TRIM genes upon P. aeruginosa and Chlamydia spp.infections in human cell lines and in mice and found both similarities and differences.The data obtained allowed us to make an assumption about the general mechanisms of the TRIM genes' work during these infections, as well as propose specific TRIM genes that may be involved in the antibacterial response.

Results
In our study, we examined both the local and systemic responses of TRIM genes to P. aeruginosa or Chlamydia spp.infections in models in vitro (human cell lines) and in vivo (DBA/2 mice), compared with non-infected controls, using RT-qPCR analysis.We considered the A549 cell line (human lung carcinoma), PC-3 cells (human prostatic adenocarcinoma), or mouse lungs as models of the primary sites of infection.The monocytic U937 cell line served as a model of the systemic immune response, and the axillary lymph nodes represented the site of adaptive immunity.The expression of several TRIM genes independently of the infections was below the level of detection in the selected cell lines or in mouse organs.In addition, not all homologue genes were found in both humans and mice.Therefore, the data were not presented for all 75 genes in the TRIM family.The expression was observed for 64, 51, and 53 TRIM genes in A549, U937, and PC-3 cell lines, respectively.Specifically, 57 TRIM genes were analyzed in the lungs and 52 TRIM genes in the lymph nodes of DBA/2 mice after infections compared to intact animals.We considered the gene up-regulated or down-regulated when its expression changed by more than 1.5 times.

The Expression
Profiles of TRIM Genes in Human A549 and U937 Cell Lines and in DBA/2 Mouse Organs after P. aeruginosa Infection Pseudomonas aeruginosa causes different severe infections, including pneumonia.We chose the A549 cells as the lung cell model and the monocytic U937 cell line for studying the non-specific immune response.To gain insights into the early response to P. aeruginosa, the analysis was conducted at 0.5 h and 1 h post infection (p.i.).We observed a massive increase in the TRIM gene expression in the A549 cell line upon P. aeruginosa infection (33% of up-regulated genes vs. 14% of the down-regulated genes) (Figure 1A).At the same time, in the U937 cell line, P. aeruginosa caused an increase in the expression of 43% of the TRIM genes 0.5 h p.i. and a decrease in the expression of 59% of the TRIM genes 1 h p.i. (Figure 1A).Presumably, the initial activation of certain TRIM genes was followed by massive suppression in the monocyte cell line.The changes in TRIM gene expression in human cell lines and in mouse organs after P. aeruginosa infection.Heatmaps of the TRIM gene expression in (A) A549 and U937 cell lines 0.5 h and 1 h p.i., and (B) mouse lungs and lymph nodes 48 h p.i. Data are presented as lgFC (gene expression p.i./gene expression in the control cells or organs).Red color: up-regulation; blue color: down-regulation.The percentage is indicated for genes with FC > 1.5 (red arrows: up-regulated genes; blue arrows: down-regulated genes).Ne: no expression.
Further, we found out what happened to the TRIM gene expression in vivo in mice after infection with P. aeruginosa.The expression level of most of the TRIM genes tended to increase in the lungs (72%) and decrease in the lymph nodes (85%) of mice infected with P. aeruginosa in comparison with intact (control) mice (Figure 1B).Therefore, upregulation of the TRIM genes was noticed both in human A549 cells and in mouse lungs after P. aeruginosa (PA) infection (a pairwise comparison of the expression profiles showed no significant difference: PA lungs vs. PA A549 at 1 h p.i., p = 0.498), while the expression of many TRIMs was decreased in human U937 cells at 1 h p.i. and in mouse lymph nodes at 48 h p.i. (PA lymph nodes vs. PA U937, p = 0.944).
Since not all the changes in the TRIM gene expression were significant, we chose for further analysis those for which p < 0.05 (Tables 1 and 2).  1 The TRIM genes, which are characteristic of each group, are highlighted in gray. 2 Unique genes for each group are in bold.
We noted similarities between the in vitro and in vivo TRIM gene transcription changes in both A549 vs. mouse lungs and U937 vs. mouse lymph nodes cases.As for mouse lung and human lung epithelial cells, TRIM16, 17, 18, 19, 20, and 21 were up-regulated in both models after P. aeruginosa infection, and TRIM7 expression was decreased in both models, as well as in mouse lymph nodes.The expression of TRIM12, 13, 14, 17, 21, 26, 27, and 56 genes appeared to be increased in the lungs and decreased in the lymph nodes.Interestingly, TRIM63 was up-regulated in A549 cells; however, its homologue was down-regulated in mouse lungs.TRIM59 and 65 appeared to be down-regulated in the monocytes and mouse lymph nodes.

The Expression Profiles of TRIM Genes in Human A549, U937, and PC-3 Cell Lines and in DBA/2 Mouse Organs after Chlamydia spp. Infection
To investigate the expression of TRIMs in Chlamydia spp.infection, we chose three representatives of the pathogen.For human cell lines, we used C. pneumoniae, which is an etiological agent of up to 20% of community-acquired atypical pneumonia, bronchitis, and upper respiratory tract infections, and C. trachomatis, the most common bacterial pathogen that causes acute and chronic infections of the reproductive organs, leading to infertility, pregnancy pathology, and infections in newborns.For the mouse infection, we used C. muridarum, a murine obligate intracellular pathogen that is widely included in mouse models of chlamydial infections of the respiratory and genital tract [13].Since Chlamydia spp.infection progresses more slowly compared to P. aeruginosa invasion, we chose longer periods of time until the moment of analysis: 4 and 8 h p.i. in cell lines and 72 h p.i. in mice.
In the A549 cell line, we observed a massive increase in TRIM gene expression (58% of the examined genes) upon C. pneumoniae infection; likewise, in U937, the number of genes with increased or decreased expression turned out to be approximately the same (in total, 27% and 29% of the genes analyzed, respectively) (Figure 2A).Only 23% of the genes were up-regulated after pulmonary C. muridarum infection in mouse lungs, whereas the expression of 44% of genes was decreased.Also, a massive decrease in TRIM gene expression in the lymph nodes was observed after C. muridarum infection (67%) (Figure 2B).Notably, TRIM8, 14, 15, 19, 20, 21, 56, and 68 were up-regulated in both the A549 cells and mouse lungs after pulmonary Chlamydia spp.infections (Table 1).Only TRIM67 was down-regulated in both models (Table 2).Interestingly, there were no compatible changes in U937 and the lymph nodes after pulmonary Chlamydia spp.infections, as was detected after P. aeruginosa infection (Tables 1 and 2).As for TRIM8, its expression was increased in the U937 cells and decreased in the mouse lymph nodes (Tables 1 and 2).It should be pointed out that TRIM7 was down-regulated in both the lungs and lymph nodes after infection with C. muridarum and P. aeruginosa.
Next, we considered the prostatic adenocarcinoma cell line PC-3 as an adequate model for C. trachomatis infection, which usually causes urogenital pathology [14].We observed an increase in the expression of 58% of the TRIM genes in infected PC-3 (Figure 2C), as in A549 upon C. pneumoniae infection (CT PC-3 vs. CP A549, 4 h p.i., p = 0.973, 8 h p.i., p = 0.902).In U937, the expression of a large part of the TRIM genes was also increased (45% of genes) (Figure 2C), in contrast to gene expression in C. pneumoniae infection.
Overall, here are some promising results generalized for both infections and both models (Tables 1 and 2).It should be noted that the pulmonary infections caused by P. aeruginosa, as well as C. pneumoniae, led to increased expression of a large pool of the same TRIM genes both in the A549 cell line and in mouse lungs (Table 1) (a pairwise comparison of the expression profiles showed no significant difference: PA lungs vs. PA A549 1 h p.i., p = 0.498; PA A549 0.5 h p.i. vs. CP A549 4 h p.i., p = 0.213; PA A549 0.5 h p.i. vs. CP A549 8 h p.i., p = 0.454; PA A549 1 h p.i. vs. CP A549 4 h p.i., p = 0.8; PA A549 1 h p.i. vs. CP A549 8 h p.i., p = 0.958).On the contrary, in the mouse lymph nodes after both infections, many of the same TRIM genes were down-regulated, demonstrating similar mechanisms of the immune response.At the same time, some of the genes were unique for each infection and cell type (highlighted in bold in Tables 1 and 2).
Summarizing, several genes were up-regulated in the cell lines that we considered to be a model of the primary site of infection: TRIM16, 47 in A549 and PC-3 after P. aeruginosa and Chlamydia spp.infections; and TRIM18, 19, 20, 21, 63 in A549 cells in response to both pulmonary infections.Several of these genes, namely TRIM16, 17, 18, 19, 20, 21, were also up-regulated in mouse lungs after P. aeruginosa, and three of them, TRIM19, 20, 21, after both pulmonary infections (P.aeruginosa and C. pneumoniae) (Table 1).We consider these genes to participate in the early non-specific immune response, which is activated at the point of contact with various bacteria.TRIM8, 14, and 68 were increased in U937 cells in response to all the pathogens examined, as well as in mouse lungs upon both infections, and in A549 after C. pneumoniae infection (Table 1).TRIM7 and 63 were down-regulated in mouse lungs upon P. aeruginosa and C. muridarum infections.Most interestingly, TRIM7 expression decreased in the lungs and in the lymph nodes after both infections.TRIM65 was down-regulated in U937 cells after P. aeruginosa and C. trachomatis infection, in mouse lungs and lymph nodes after C. muridarum infection, and in lymph nodes upon P. aeruginosa infection (Table 2).In addition, we found that the dynamics of TRIM63 expression during both bacterial infections in mice (in the lungs, Table 2) was opposite to that in humans (in A549 cells, Table 1), which means that the mouse model is not suitable for studying the role of TRIM63 in human bacterial-caused diseases.In order to determine the possible pathway of bacteria's influence on the TRIM genes, we compared our results with those in recent studies where the expression of TRIM genes upon Toll-like receptor (TLR) activation in THP1-derived macrophages [15], or after interferon (IFN) type I or II stimulation in primary monocyte-derived macrophages, or peripheral blood lymphocytes [16] was analyzed.We found that the majority of the TRIMs that were activated after P. aeruginosa or Chlamydia spp.infections (TRIM5, 13,14,15,18,19,20,21,22,25,26,31,35,36,37,50,55,56,61,63,65, 69, and 71) were up-regulated following TLR stimulation and/or by IFNs (Table S2), while TRIM59 and TRIM66 were down-regulated under these conditions and upon P. aeruginosa infection (Table S2).
Likewise, we found that several TRIMs (TRIM 13, 14, 15, 18, 19, 20, 21, 25, 26, and 56) activated in P. aeruginosa or C. muridarum infections were also up-regulated following TLR activation and/or stimulation with IFNs (Table S3).Notably, TRIM8, 16, 17, and 27 transcription was increased and the expression of TRIM65 and 67 was decreased in the response to various pathogens in different human cell lines and in mouse lungs as well (Tables S2 and S3), suggesting a universal antibacterial mechanism for these genes in mice and humans.
Additionally, we performed bioinformatic analysis of the RNA-seq data on transcriptomes of mouse lung after P. aeruginosa (24 h p.i.) and C. muridarum (7 days p.i.) infections published by Ebenezer et al. (2019) [17] and Virok et al. ( 2019) [18] and demonstrated the same trends in the expression of TRIM genes, taking into account the difference in the time of sampling (Table S3).

Western Blot Protein Assay of Several TRIMs of Interest in the U937 Cell Line
Next, we selected three TRIM genes, namely TRIM8, 14, and 17, to study changes in the expression of the corresponding proteins in cell line U937 after infection with P. aeruginosa, C. pneumoniae, and C. trachomatis.Western blot analysis showed changes in TRIM8 and TRIM14 protein expression, confirming the involvement of the proteins in the immune response to the studied bacterial infections (Figure 3).Expression of the TRIM17 protein remained practically unchanged, as was its transcription.(Figure 3).

The Expression Profiles of IFNs and Inflammatory
Genes in Human A549, U937, and PC-3 Cell Lines, and in DBA/2 Mouse Organs after P. aeruginosa and Chlamydia spp.Infections Since TRIM genes are known to be interferon-stimulated genes (ISGs) and also to participate in the inflammatory response, we analyzed type I IFNs (IFNA, B), a type II IFN (IFNG), and pro-inflammatory gene (TNFA, IL1B, and IL6) expression using RT-qPCR, both in vitro and in vivo (Figure 4).

A549
Mouse  The IFN system was mostly involved in response to both types of infection in mouse lungs and in U937 cells, while in A549, the transcription of IFNs remained unchanged or slightly increased (the expression of IFNG was even decreased at 4 h p.i. with C. pneumoniae) (Figure 4).Importantly, TNFA, IL1B, and IL6 were up-regulated in response to the bacterial infections in both epithelial A549 and monocyte U937 cells, as well as in vivo in the lungs (Figure 4).Additionally, a significant increase in the expression of pro-inflammatory genes (TNFA, IL1B, and IL6) in PC-3 cells infected with C. trachomatis was reported earlier [14], which was in line with our observations.However, in mouse lymph nodes, genes encoding both the IFNs and pro-inflammatory cytokines were suppressed after P. aeruginosa infection.TNFA, IL1B, and IL6 gene expression dropped when mice were infected with C. muridarum, while IFNB and IFNG expression slightly increased (Figure 4).Given that the lymph nodes are responsible for adaptive immunity that becomes detectable within the lymph nodes after 5 days p.i. [19,20], we assume that the IFN and pro-inflammatory gene programs were not yet activated in the lymph nodes in our models of the early stages of acute infections.
Thus, it can be supposed that the described changes in the expression of the TRIM genes after infection may be due to the TLRs activation by bacterial molecular patterns and cannot be explained by the activation of only interferon signaling.

Discussion
Throughout life, we constantly encounter infections caused by various pathogens.The formation of an effectively protective immune response depends on the functioning of many adapter molecules involved in the transmission, as well as the amplification or attenuation, of the signal from receptors, which meet pathogens, to the transcription factors in eukaryotic cells.Among currently known adapter molecules, proteins from the TRIM family are of particular interest since they are shown to be actively involved in the antiviral response of innate immunity [2,21].However, there is still very little information regarding the role of TRIM proteins in the antibacterial response.In our opinion, new findings in this field will contribute to a more comprehensive understanding of intracellular signaling cascades involved in the immune response.
P. aeruginosa and Chlamydia spp.are pathogens that can successfully fight the immune system [11,12], due to the high level of antibiotic resistance and tolerance to the action of many antibacterial drugs, inherent in these pathogens.This determines the need for fundamental studies on the molecular mechanisms of the interaction of these pathogens with the host's innate immune system and the search for new approaches to combat diseases caused by these extremely problematic pathogens.
Our study showed similarities and differences in expression for a number of TRIM genes after P. aeruginosa and Chlamydia spp.Infections, both in vitro (human cell lines A549, U937, and PC-3) and in vivo (lungs and lymph nodes of mice) (Figure 5).Activation and deactivation of the TRIM genes occurred quite synchronously, depending on the types of cells and pathogens.Namely, two different pathogens, P. aeruginosa and C. pneumoniae, caused an increase in TRIM expression in A549 cells and in mouse lungs.We assumed that such a correspondence might be due to the fact that the lungs were a primary focus for both infections in vivo and that A549 cells are related to lung tissue due to the cells' nature.In the same way, mass up-regulation of the TRIM genes was found in the prostatic adenocarcinoma cell line PC-3 after C. trachomatis infection, which causes damage to the genital tract.Thus, we suggest that the expression of TRIM genes apparently depends on whether the cells belong to the primary site of infection or not.Strikingly, we also observed the massive suppression of TRIM gene expression in the lymph nodes after infections with P. aeruginosa and C. muridarum.On the one hand, that could be explained by the fact that during acute infection, activation of the adaptive immunity in the lymph nodes is delayed.On the other hand, such a suppression of TRIMs and even of non-specific pro-inflammatory genes (TNFA, IL1B, and IL6) might be a result of the active influence of pathogens on the host's immune system.It should be mentioned that several TRIM genes, whose expression was decreased in the lymph nodes after bacterial infections, were previously reported to take part in T-cell signaling (TRIM21, 27, 28, 32, 33) [22].It is possible that bacteria, counteracting the immune defense, suppress the activation of this group of TRIM genes.
Additionally, our data on the down-regulation of TRIM17, 21,27,32,35,45,46,47,56, and 65 in the mouse lymph nodes correlate with those obtained by Chen et al. [3] in the blood of tuberculosis patients and/or in macrophages after infection with Mycobacterium smegmatis [3].
Activation of the pro-inflammatory response is often the result of stimulation of TLRs, which are the main receptors in the innate immune system and are able to recognize pathogens of different natures depending on the type of receptor.There is evidence that P. aeruginosaor Chlamydia-derived products are ligands for different TLRs [23][24][25].TLR4 can specifically recognize gram-negative bacterial LPS and use both MyD88 and TRIF adaptor proteins, leading to the activation of genes encoding pro-inflammatory cytokines and interferons [26].At the same time, many TRIM genes are IFN-inducible in the response to infections [16,27,28] and, in turn, can activate IFN production [2].Our comparison of the TRIM gene expression changes upon P. aeruginosa or Chlamydia spp.infections with known data concerning the regulation of TRIM genes under stimulation of TLRs [15] or treatment by IFNs [16] revealed similar activation of many TRIM genes (TRIM5, 6, 10, 13,14,15,18,19,20,21,22,25,26,31,34,35,36,37,50,55,61,63,65,69, 71 (in A549), 56, 58 (in mouse lungs)) (Table S2 and S3).The expression of other TRIM genes was reduced in our study (mainly in the lymph nodes of mice), which was consistent with the corresponding data on monocyte derived macrophages or peripheral blood lymphocytes under IFN treatment [16], or under TLR stimulation [15] (TRIM28, 32, 37, 41, 55 (in lungs), 59, 61, 66 (in U937) (Tables S2 and S3).
Our analysis, however, showed that although the IFNs were up-regulated in infected U937 monocytes and in the lungs of mice, they remained unchanged or only slightly increased in A549 cells upon both infections and were dramatically down-regulated in mouse lymph nodes under P. aeruginosa infection, suggesting their rather limited role in the bacterial infections we studied.The known literature data showing the involvement of many TRIM genes in inflammation [7,27,[29][30][31][32][33][34][35][36][37][38] is supported by our data.Pro-inflammatory genes were found to be activated in all human cell lines, as well as in mouse lungs, after both pathogen infections.The exception was only mouse lymph nodes, especially after P. aeruginosa infection, where their expression dropped, which was also observed for the IFNs and for the majority of TRIM genes, as mentioned above.Interestingly, one of these genes with decreased expression is TRIM72, which was demonstrated to promote P. aeruginosa-induced inflammation in mouse lungs [39].This gene was shown to act via the complement receptor (CR) in the Ig superfamily (CRIg) in alveolar macrophages [39].This fact indicates the existence of various mechanisms for the participation of TRIM genes in the immune response to bacterial infections, not only through TLRs but also through other receptors.
It should be noted that the expression of the TRIM63 gene changed in the opposite way in human cells and in mouse lungs.Therefore, caution should be exercised when extrapolating mouse data to humans.
Summarizing, our data show that bacterial pathogens directly influence signaling mechanisms of innate immunity, including a network of TRIM family genes (Figure 5).For the first time, we have compared the effects of two different pathogens in human cell lines (model system) and in animals (organism level) on the expression of TRIM family genes, as well as genes encoding interferons and pro-inflammatory factors.Among the identified genes, there are a number of TRIM genes that are involved in the overall immune response, both in vitro in human cells and in vivo in mouse organs, when infected with two different types of pathogens (TRIM8, 14,16,17,18,19,20,21,47,68).Along with this, TRIM genes specific for both the pathogen and the organism were found (e.g., TRIM59 upon P. aeruginosa infection in human U937 and mouse lymph nodes; TRIM67 in mouse lungs and lymph nodes, as well as in A549 after Chlamydia spp.infections).The TRIM7 gene was the only gene whose expression was decreased in mouse lungs and lymph nodes after both infections and in A549 after P. aeruginosa infection.Further study on the role of the TRIM genes, identified in our work, involved in the immune response to P. aeruginosa and Chlamydia spp.will allow us to determine, in more detail, the mechanisms of the formation of the antibacterial immune response to these pathogens.
For measuring C. trachomatis and C. pneumoniae infectivity, confluent daily A549, PC-3 cell monolayers, or U-937 suspension cultures were infected with 10-fold dilutions of suspension of purified EB in 24-well plates (Corning Inc., Corning NY, USA), with 12 mm round cover glasses (Menzel, Berlin, Germany).After 48 h of incubation (5% CO 2 , 37 • C), the glasses were washed with 0.1 mM PBS, dried in air, and fixed in cold acetone for 15 min at room temperature (RT).The U-937 cells were precipitated by centrifugation for 10 min at 500 rpm, washed with 0.1 mM PBS, and fixed with ice-cold acetone for 15 min at RT.
The preparations were stained with monoclonal FITC-labeled antibodies for the species-specific protein antigen of C. trachomatis (Bio-Rad, Hercules, CA, USA) and for the genus-specific antigen of bacterial lipopolysaccharide to detect the antigen of C. pneumoniae (CABT-RM310, Creative Diagnostics, Shirley, NY, USA).The monolayer was examined by luminescent microscopy in a Nikon Eclipse Ni-U luminescent microscope (eyepieces 1.3, lens ×40).The percentage of infected cells in 30 independent visual fields was calculated, and the CFU per 1 mL was determined [43].
The 10-fold dilutions of a suspension from a night culture of P. aeruginosa KB-6 grown in LB broth were carried out, followed by seeding on cetrimide agar.Cultures were cultivated for 24 h at 37 • C. The count of the grown colonies of P. aeruginosa was carried out, and the CFU/mL was determined [40].

Infection of the Cell Lines
We determined the required multiplicity of infections: C. trachomatis-5 MOI, C. pneumoniae-10 MOI, Pseudomonas aeruginosa-5 MOI.After the addition of the infectious material, the culture plates were centrifuged at 3000 rpm for 60 min at 25 • C, and the cells were incubated at 5% CO 2 at 37 • C. Further, the cell monolayer was washed with PBS and then lysed with TRIzol (Thermo Fisher Scientific, Waltham, MA, USA).Cell lysates were collected at 4 and 8 h after Chlamydia spp.infection, and at 0.5 and 1 h after P. aeruginosa infection in two duplicate experiments.The samples were stored at −70 • C.

Mouse Infection
The study was conducted in compliance with the Guide for the Care and Use of Laboratory Animals (NIH Publication #85-23, revised 1996) and with the recommendations in the national guidelines and was approved by the Gamaleya National Research Center Animal Care Committee (protocol #19, 2 July 2020).
The mice were anesthetized with inhalational diethyl ether (Ecos-1, Moscow, Russia) and injected intranasally with 40 µL per mouse via saline containing 5 × 10 5 CFU of the C. muridarum Nigg strain, or 10 7 CFU of Pseudomonas aeruginosa.After infection, the animals were monitored twice a day.During the accumulation of the pathogen in the lungs and the development of pneumonia, the condition of the mice worsened: food refusal; sticky hair; rapid, shallow breathing; and low activity appeared.On the second (P.aeruginosa) or third (C.muridarum) day after infection, the animals were subjected to euthanasia and subsequent autopsy.The lungs and axillary lymph nodes were taken from the mice in all the groups.The organs were treated with 1.0 mL of TRIzol (Thermo Fisher Scientific, Waltham, MA, USA), homogenized, and stored at -70 • C.

Quantitative PCR
Total RNA was extracted from the cells or organs using a TRIzol RNA purification kit (Thermo Fisher Scientific, Waltham, MA, USA), as recommended by the manufacturer, with subsequent DNase treatment (Thermo Fisher Scientific, Waltham, MA, USA).cDNA was synthesized from 2 µg of total RNA using the M-MLV reverse transcriptase (Evrogen, Moscow, Russia) with random primers.The obtained cDNA was amplified using a Light-Cycler 96 instrument (Roche, Basel, Switzerland).The reaction conditions were as follows: denaturation at 95 • C (3 min), followed by 40 cycles (95 • C, 15 s; 55-65 • C, 20 s; and 72 • C, 45 s).The reaction mixture qPCRmix-HS SYBR (Evrogen, Moscow, Russia) was used.As a reference gene, the 18S rRNA was used.Relative changes in the gene expression levels were determined using the 2 −∆∆Ct method [45].The utilized primer sequences are given in Table S1.
Figure 1.The changes in TRIM gene expression in human cell lines and in mouse organs after P. aeruginosa infection.Heatmaps of the TRIM gene expression in (A) A549 and U937 cell lines 0.5 h and 1 h p.i., and (B) mouse lungs and lymph nodes 48 h p.i. Data are presented as lgFC (gene expression p.i./gene expression in the control cells or organs).Red color: up-regulation; blue color: down-regulation.The percentage is indicated for genes with FC > 1.5 (red arrows: up-regulated genes; blue arrows: down-regulated genes).Ne: no expression.

Figure 4 .
Figure 4. Analysis of the expression of genes encoding IFNs and pro-inflammatory proteins in human cell lines and mouse organs after P. aeruginosa and Chlamydia spp.infections.Heatmaps of the immune gene expression in the A549 and U937 cell lines and mouse lungs and lymph nodes after (A) P. aeruginosa and (B) Chlamydia spp.infections.Data are presented as lgFC (gene expression after infection/gene expression in the control cells or organs).Red color: up-regulation; blue color: down-regulation.* p < 0.05, ** p < 0.01, *** p < 0.001.
of TRIM genes (* -significant for all) Down-regulation of TRIM genes (* -significant for all) Specific for P A infection Specific for Chlamydia spp.infection

Figure 5 .
Scheme of experiment

Table 1 .
TRIM genes whose transcription was increased in human cell lines or in mouse organs after infection compared to the controls (p < 0.05).

Table 2 .
TRIM genes whose transcription was decreased in human cell lines or in mouse organs after infection compared to the controls (p < 0.05).