Hypoxia-Inducible Factor Signaling in Macrophages Promotes Lymphangiogenesis in Leishmania major Infection.

Vascular remodeling is a phenomenon seen in the cutaneous lesions formed during infection with Leishmania parasites. Within the lesion, Leishmania major infection leads to the infiltration of inflammatory cells, including macrophages, and is associated with hypoxic conditions and lymphangiogenesis in the local site. This low-oxygen environment is concomitant with the expression of hypoxic inducible factors (HIFs), which initiate the expression of vascular endothelial growth factor-A (VEGF-A) in macrophages during the infection. Here, we found that macrophage hypoxia is elevated in the skin, and the HIF target Vegfa is preferentially expressed at the site of infection. Further, transcripts indicative of both HIF-1α and HIF-2α activation were increased at the site of infection. Given that HIF mediates VEGF-A and that VEGF-A/VEGFR-2 signaling induces lymphangiogenesis, we wanted to investigate the link between myeloid HIF activation and lymphangiogenesis during L. major infection. We show that myeloid aryl hydrocarbon receptor nuclear translocator (ARNT)/HIF/VEGF-A signaling promotes lymphangiogenesis (the generation of newly formed vessels within the local lymphatic network), which helps resolve the lesion by draining away inflammatory cells and fluid. Concomitant with impaired lymphangiogenesis, we find the deletion of myeloid ARNT/HIF signaling leads to an exacerbated inflammatory response associated with a heightened CD4+ Th1 immune response following L. major infection. Altogether, our data suggest that VEGF-A-mediated lymphangiogenesis occurs through myeloid ARNT/HIF activation following Leishmania major infection and this process is critical in limiting immunopathology.

Leishmania Infection Induces Macrophage Vascular Endothelial Growth Factor A Production in an ARNT/HIF-Dependent Manner.

Cutaneous leishmaniasis is characterized by vascular remodeling. Following infection with Leishmania parasites, the vascular endothelial growth factor A (VEGF-A)/VEGF receptor 2 (VEGFR-2) signaling pathway mediates lymphangiogenesis, which is critical for lesion resolution. Therefore, we investigated the cellular and molecular mediators involved in VEGF-A/VEGFR-2 signaling using a murine model of infection. We found that macrophages are the predominant cell type expressing VEGF-A during Leishmania major infection. Given that Leishmania parasites activate hypoxia-inducible factor 1α (HIF-1α) and this transcription factor can drive VEGF-A expression, we analyzed the expression of HIF-1α during infection. We showed that macrophages were also the major cell type expressing HIF-1α during infection and that infection-induced VEGF-A production is mediated by ARNT/HIF activation. Furthermore, mice deficient in myeloid ARNT/HIF signaling exhibited larger lesions without differences in parasite numbers. These data show that L. major infection induces macrophage VEGF-A production in an ARNT/HIF-dependent manner and suggest that ARNT/HIF signaling may limit inflammation by promoting VEGF-A production and, thus, lymphangiogenesis during infection.

Neonatal Diet Impacts Bioregional Microbiota Composition in Piglets Fed Human Breast Milk or Infant Formula.

BACKGROUND: Early infant diet influences postnatal gut microbial development, which in turn can modulate the developing immune system. OBJECTIVES: The aim of this study was to characterize diet-specific bioregional microbiota differences in piglets fed either human breast milk (HM) or infant formula. METHODS: Male piglets (White Dutch Landrace Duroc) were raised on HM or cow milk formula (MF) from postnatal day (PND) 2 to PND 21 and weaned to an ad libitum diet until PND 51. Piglets were euthanized on either PND 21 or PND 51, and the gastrointestinal contents were collected for 16s RNA sequencing. Data were analyzed using the Quantitative Insight into Microbial Ecology. Diversity measurements (Chao1 and Shannon) and the Wald test were used to determine relative abundance. RESULTS: At PND 21, the ileal luminal region of HM-fed piglets showed lower Chao1 operational taxonomic unit diversity, while Shannon diversity was lower in cecal, proximal colon (PC), and distal colon (DC) luminal regions, relative to MF-fed piglets. In addition, at PND 51, the HM-fed piglets had lower genera diversity within the jejunum, ileum, PC, and DC luminal regions, relative to MF-fed piglets. At PND 21, Turicibacter was 4- to 5-fold lower in the HM-fed piglets' ileal, cecal, PC, and DC luminal regions, relative to the MF-fed piglets. Campylobacter is 3- to 6-fold higher in HM-fed piglets duodenal, ileal, cecal, PC, and DC luminal regions, in comparison to MF-fed piglets. Furthermore, the large intestine (cecum, PC, and rectum) luminal region of HM-fed piglets showed 4- to 7-fold higher genera that belong to class Bacteroidia, in comparison to MF-fed piglets at PND 21. In addition, at PND 51 distal colon lumen of HM-fed piglets showed 1.5-fold higher genera from class Bacteroidia than the MF-fed piglets. CONCLUSIONS: In the large intestinal regions (cecum, PC, and rectum), MF diet alters microbiota composition, relative to HM diet, with sustained effects after weaning from the neonatal diet. These microbiota changes could impact immune system and health outcomes later in life.

Human Breast-Milk Feeding Enhances the Humoral and Cell-Mediated Immune Response in Neonatal Piglets.

Background: The benefits of breastfeeding infants are well characterized, including those on the immune system. However, determining the mechanism by which human breast milk (HBM) elicits effects on immune response requires investigation in an appropriate animal model. Objective: The primary aim of this study was to develop a novel porcine model and to determine the differential effects of feeding HBM and a commercial milk formula (MF) on immune response and gastrointestinal microbial colonization in a controlled environment. Methods: Male piglets were fed HBM (n = 26) or MF (n = 26) from day 2 through day 21. Piglets were vaccinated (n = 9/diet group) with cholera toxin and cholera toxin subunit B (CTB) and tetanus toxoid at 21 d or were fed placebo (n = 6/diet group) and then weaned to a standard solid diet at the age of 21 d. Humoral and cell-mediated immune responses were assessed from blood on days 35 and 48. Immune response was further examined from tissues, including mesenteric lymph nodes (MLNs), Peyer's patches (PPs), and spleen. The colonization of gut microbiota was characterized from feces on days 16 and 49. Results: Serum antibody titers in piglets fed HBM were 4-fold higher (P < 0.05) to CTB and 3-fold higher (P < 0.05) to tetanus toxoid compared with piglets fed MF on day 48. Compared with MF, the numbers of immunoglobulin A antibody-producing cells to CTB were 13-fold higher (P < 0.05) in MLNs and 11-fold higher (P < 0.05) in PPs in the HBM diet group on day 51. In addition, significantly increased T cell proliferation was observed in the HBM group relative to the MF group. Furthermore, microbial diversity in the HBM group was lower (P < 0.05) than in the MF group. Conclusions: This porcine model appears to be valid for studying the effects of early postnatal diet on immune responses and the gastrointestinal microbiome. Our results lay the groundwork for future studies defining the role of infant diet on microbiota and immune function.

Early Postnatal Diets Affect the Bioregional Small Intestine Microbiome and Ileal Metabolome in Neonatal Pigs.

Background: Breastfeeding is known to be protective against gastrointestinal disorders and may modify gut development. Although the gut microbiome has been implicated, little is known about how early diet affects the small intestine microbiome.Objective: We hypothesized that disparate early diets would promote unique microbial profiles in the small intestines of neonatal pigs.Methods: Male and female 2-d-old White Dutch Landrace pigs were either sow fed or provided dairy (Similac Advance powder; Ross Products Abbott Laboratories) or soy (Enfamil Prosobee Lipil powder; Mead Johnson Nutritionals) infant formulas until day 21. Bacterial ecology was assessed in the contents of the small intestine through the use of 16S ribosomal RNA sequencing. α-Diversity, ß-diversity, and differential abundances of operational taxonomic units were assessed by ANOVA, permutational ANOVA, and negative binomial regression, respectively. Ileum tissue metabolomics were measured by LC-mass spectrometry and assessed by weighted correlation network analysis.Results: Greater α-diversity was observed in the duodena of sow-fed compared with formula-fed neonatal pigs (P < 0.05). No differences were observed in the ilea. Firmicutes represented the most abundant phylum across all diets in duodena (78.8%, 80.1%, and 53.4% relative abundance in sow, dairy, and soy groups, respectively), followed by Proteobacteria in sow (12.2%) and dairy (12.4%) groups and Cyanobacteria in soy-fed (36.2%) pigs. In contrast to those in the duodenum, Proteobacteria was the dominant phylum in the ileum, with >60% relative abundance in all of the groups. In the duodenum, 77 genera were altered by diet, followed by 48 in the jejunum and 19 in the ileum. Metabolomics analyses revealed associations between ileum tissue metabolites (e.g., acylcarnitines, 3-aminoisobutyric acid) and diet-responsive microbial genera.Conclusions: These results indicate that the neonatal diet has regional effects on the small intestine microbiome in pigs, with the most pronounced effects occurring in the duodena. Regional effects may be important factors when considering gut tissue metabolism and development in the postnatal period.

Formula diet alters small intestine morphology, microbial abundance and reduces VE-cadherin and IL-10 expression in neonatal porcine model.

BACKGROUND: Breastfeeding is associated with a variety of positive health outcomes in children and is recommended exclusively for the first 6 months of life; however, 50-70 % of infants in the US are formula-fed. To test the hypothesis that immune system development and function in neonates and infants are significantly influenced by diet, 2-day old piglets were fed soy or milk formula (n = 6/group/gender) until day 21 and compared to a sow-fed group (n = 6/gender). METHODS: Histomorphometric analyses of ileum, jejunum and Peyer's patches were carried out, to determine the inflammation status, mRNA and protein expression of pro-inflammatory, anti-inflammatory and growth-related chemokines and cytokines. RESULTS: In formula-fed animals, increases in ileum and jejunum villus height and crypt depth were observed in comparison to sow-fed animals (jejunum, p < 0.01 villus height, p < 0.04 crypt depth; ileum p < 0.001 villus height, p < 0.002 crypt depth). In formula-fed the lymphoid follicle size (p < 0.01) and germinal centers (p < 0.01) with in the Peyer's patch were significantly decreased in comparison to sow-fed, indicating less immune education. In ileum, formula diet induced significant up-regulation of AMCFII, IL-8, IL-15, VEGFA, LIF, FASL, CXCL11, CCL4, CCL25 and down-regulation of IL-6, IL-9, IL-10, IL-27, IFNA4, CSF3, LOC100152038, and LOC100736831 at the transcript level. We have confirmed some of the mRNA data by measuring protein, and significant down-regulation of anti-inflammatory molecule IL-10 in comparison to sow-fed piglets was observed. To further determine the membrane protein expression in the ileum, VE-cadherin, occludin, and claudin-3, Western blot analyses were conducted. Sow fed piglets showed significantly more VE-Cadherin, which associated with levels of calcium, and putrescine measured. It is possible that differences in GI tract and immune development are related to shifts in the microbiome; notably, there were 5-fold higher amounts of Lactobacillaceae spp and 3 fold higher Clostridia spp in the sow fed group in comparison to milk formula-fed piglets, whereas in milk formula-fed pigs Enterobacteriaceae spp was 5-fold higher. CONCLUSION: In conclusion, formula diet alters GI morphology, microbial abundance, intestinal barrier protein VE-cadherin and anti-inflammatory molecule IL-10 expression. Further characterization of formula effects could lead to modification of infant formula to improve immune function, reduce inflammation and prevent conditions such as allergies and infections.

Chlamydial variants differ in ability to ascend the genital tract in the guinea pig model of chlamydial genital infection.

An important question in the study of chlamydial genital tract disease is why some women develop severe upper tract disease while others have mild or even "silent" infections with or without pathology. Animal studies suggest that the pathological outcome of an infection is dependent upon both the composition of the infecting chlamydial population and the genotype of the host, along with host physiological effects, such as the cyclical production of reproductive hormones and even the size of the infecting inoculum or the number of repeated infections. In this study, we compared two variants of Chlamydia caviae, contrasting in virulence, with respect to their abilities to ascend the guinea pig genital tract. We then determined the effect of combining the two variants on the course of infection and on the bacterial loads of the two variants in the genital tract. Although the variants individually had similar infection kinetics in the cervix, SP6, the virulent variant, could be isolated from the oviducts more often and in greater numbers than the attenuated variant, AZ2. SP6 also elicited higher levels of interleukin 8 (IL-8) in the lower genital tract and increased leukocyte infiltration in the cervix and uterus compared to AZ2. When the two variants were combined in a mixed infection, SP6 outcompeted AZ2 in the lower genital tract; however, AZ2 was able to ascend the genital tract as readily as SP6. These data suggest that the ability of SP6 to elicit an inflammatory response in the lower genital tract facilitates the spread of both variants to the oviducts.

Enhanced translational activity is linked to lymphatic endothelial cell activation in cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) is a significant public health problem leading to permanently disfiguring skin lesions caused by Leishmania parasites. Lesion severity stems from an excessive host inflammatory response that prevents healing. Here, we characterized the transcriptional and translational responses of lymphatic endothelial cells (LECs) during murine CL using historical single-cell RNA sequencing data combined with flow cytometry and in vivo puromycin incorporation to assess translational activity. We identified upregulation of antigen presentation pathways including MHC-I, MHC-II, and immunoproteasome transcripts in dermal LECs from Leishmania major -infected mice compared to naive controls. LECs also exhibited increased expression of guanylate binding proteins and interferon-inducible genes, indicative of immune activation. Moreover, our findings demonstrate that LECs in leishmanial lesions displayed heightened translational activity relative to LECs from uninflamed ears, and LEC translational activity was highest in activated LECs. Furthermore, LEC translational activity exceeded that of other cell types within the lesion microenvironment. Validating the transcriptomic data, LECs in lesions expressed elevated MHC-II and programmed death-ligand 1 (PDL-1), supporting their potential role in antigen presentation. Functional assays using DQ-OVA confirmed that LECs from leishmanial lesions efficiently uptake and process antigens, highlighting their capability as antigen presenting cells in the inflamed dermal microenvironment. Overall, our study reveals the activation status of LECs in leishmanial lesions, shedding light on their potential role in shaping local immunity and inflammation in a variety of skin diseases.

HIF-α signaling regulates the macrophage inflammatory response during Leishmania major infection.

Cutaneous leishmaniasis (CL) contributes significantly to the global burden of neglected tropical diseases, with 12 million people currently infected with Leishmania parasites. CL encompasses a range of disease manifestations, from self-healing skin lesions to permanent disfigurations. Currently there is no vaccine available, and many patients are refractory to treatment, emphasizing the need for new therapeutic targets. Previous work demonstrated macrophage HIF-α-mediated lymphangiogenesis is necessary to achieve efficient wound resolution during murine L. major infection. Here, we investigate the role of macrophage HIF-α signaling independent of lymphangiogenesis. We sought to determine the relative contributions of the parasite and the host-mediated inflammation in the lesional microenvironment to myeloid HIF-α signaling. Because HIF-α activation can be detected in infected and bystander macrophages in leishmanial lesions, we hypothesize it is the host's inflammatory response and microenvironment, rather than the parasite, that triggers HIF-α activation. To address this, macrophages from mice with intact HIF-α signaling (LysM Cre ARNT f/+ ) or mice with deleted HIF-α signaling (LysM Cre ARNT f/f ) were subjected to RNASequencing after L. major infection and under pro-inflammatory stimulus. We report that L. major infection alone is enough to induce some minor HIF-α-dependent transcriptomic changes, while infection with L. major in combincation with pro-inflammatory stimuli induces numerous transcriptomic changes that are both dependent and independent of HIF-α signaling. Additionally, by coupling transcriptomic analysis with several pathway analyses, we found HIF-α suppresses pathways involved in protein translation during L. major infection in a pro-inflammatory environment. Together these findings show L. major induces a HIF-α-dependent transcriptomic program, but HIF-α only suppresses protein translation in a pro-inflammatory environment. Thus, this work indicates the host inflammatory response, rather than the parasite, largely contributes to myeloid HIF-α signaling during Leishmania infection.

Synthesis and in vitro evaluation shows disquaramide compounds are a promising class of anti-leishmanial drugs.

An increasing number of treatment failures with current pharmaceutics, as well as a lack of a vaccine, demonstrates the need to develop new treatment options for leishmaniasis. Herein, we describe the synthesis and in vitro analysis of 24 disquaramide compounds targeting the Leishmania major parasite. Of the compounds that were evaluated, six of them ( 13 , 19 , 20 , 22 , 24 , and 26 ) were capable of significantly decreasing the number of parasites by up to 42% compared to the control by day four. This demonstrates that disquaramides either impair parasite replication or have leishmancidal effects. Additionally, none of the disquaramide compounds tested displayed host cell cytotoxicity. These experiments provide evidence that disquaramides have the potential to be effective anti-leishmanial therapeutics.

Differential susceptibilities to azithromycin treatment of chlamydial infection in the gastrointestinal tract and cervix.

Evidence from animal studies suggests that chlamydiae may persist in the gastrointestinal tract (GI) and be a reservoir for reinfection of the genital tract. We hypothesize that there may be a differential susceptibility of organisms in the GI and genital tracts. To determine the effect of azithromycin on persistent chlamydial gut infection, C57BL/6 and BALB/c mice were infected orally and genitally and treated with azithromycin (Az) orally (20, 40, or 80 mg/kg of body weight), and the numbers of chlamydiae were determined from cervix and cecal tissues. The Az concentration in the cecum and cervix was measured by high-performance liquid chromatography with electrochemical detection (HPLC-ECD). Az treatment cleared genital infection in both C57BL/6 and BALB/c mice; however, GI infection was not cleared with the same doses. HPLC data showed the presence of Az at both sites of infection, and significant amounts of Az were measured in treatment groups. However, no significant difference in Az levels between the cecum and the cervix was observed, indicating similar levels of Az reaching both sites of infection. These data indicate that antibiotic levels that are sufficient to cure genital infection are ineffectual against GI infection. The results suggest a reevaluation of antibiotic therapy for chlamydial infection.

BOTH THE INFECTION STATUS AND INFLAMMATORY MICROENVIRONMENT INDUCE TRANSCRIPTIONAL REMODELING IN MACROPHAGES IN MURINE LEISHMANIAL LESIONS.

Cutaneous leishmaniasis is caused by infection with the protozoan parasite Leishmania, which resides intracellularly in dermal macrophages (Mø), producing lesions. The skin lesions are characterized by proinflammatory cytokines and growth factors as well as inflammatory hypoxia, creating a stressful microenvironment for Mø. Of importance, not all Mø in lesions harbor parasites. To distinguish the influence of the parasite from the inflammatory microenvironment after Leishmania major (LM) infection on the Mø, we performed single-cell RNA sequencing and compared Mø associated with LM transcripts (or 'infected' Mø) with Mø not associated with LM transcripts (or 'bystander' Mø) within the lesions. Our findings show coordinated lysosomal expression and regulation signaling with increased cathepsin and H+-ATPase transcripts are upregulated in infected compared with bystander Mø. Additionally, eukaryotic initiation factor 2 (EIF2) signaling is downregulated in infected compared with bystander Mø, which includes many small and large ribosomal subunit (Rps and Rpl) transcripts being decreased in Mø harboring parasites. Furthermore, we also find EIF2 signaling including EIF, Rps, and Rpl transcripts being downregulated in bystander Mø compared with Mø from naïve skin. These data suggest that both the parasite and the inflammatory host microenvironment affect the transcription of ribosomal machinery in lesional Mø, thereby potentially affecting the ability of these cells to perform translation, protein synthesis, and thus function. Altogether, these results suggest that both the parasite and host inflammatory microenvironment independently drive transcriptional remodeling in Mø during LM infection in vivo.

Effect of inflammatory response on in vivo competition between two chlamydial variants in the guinea pig model of inclusion conjunctivitis.

In order to study the interaction of variants in in vivo infection, we employed an azithromycin-resistant mutant (AZ(2)) and its wild-type parent (SP(6)) in the guinea pig model of Chlamydia caviae conjunctival infection. When each strain was inoculated individually into conjunctiva, both attained the same level of growth, but AZ(2) elicited less pathology. However, when equal numbers of the two strains were inoculated together into the guinea pig conjunctiva, SP(6) produced a significantly greater number of inclusion-forming units than AZ(2), and the pathology reflected that of a SP(6) monoinfection. The goal of this study was to further characterize the dynamics of concomitant infection of these two distinct variants, with particular emphasis on the impact of the host response on the in vivo growth of each organism and the development of pathology. Animals infected with AZ(2) had reduced conjunctival infiltration with CD45(+) cells and neutrophils as well as a reduced interleukin-8 (IL-8) response. Gene expression of gamma interferon (IFN-γ), tumor necrosis factor alpha (TNF-α), CCL2, and CCL5 was also significantly lower in AZ(2)-infected animals. The lower inflammatory response induced by AZ(2) was associated with its decreased ability to activate NF-κB via Toll-like receptor 2 (TLR2). In general, the inflammatory response in animals infected with both variants was greater than in infection with AZ(2) alone, resulting in lower numbers of AZ(2) than those of SP(6) in the mixed infection. Our results suggest that the ability to elicit an inflammatory response is an important factor in the dynamics of mixed infection with strains that display different pathological phenotypes.

Bacterial and Fungal Adaptations in Cecum and Distal Colon of Piglets Fed With Dairy-Based Milk Formula in Comparison With Human Milk.

Exclusive breastfeeding is recommended to newborns during the first 6 months of life, whereas dairy-based infant formula is an alternative nutrition source offered to infants. Several studies demonstrated that breastfed infants have a different gut bacterial composition relative to formula-fed infants. In addition, animal models have shown that human milk (HM)-fed piglets had a distinct intestinal bacterial composition compared with milk formula (MF)-fed piglets. However, the gut fungal composition and the interactions with the bacterial community in breastfed compared with formula-fed infants remain to be investigated. In an attempt to evaluate such differences, we used an animal model to perform a shotgun metagenomics analysis on the cecal and distal colon contents of neonatal piglets fed with pasteurized HM or a dairy-based infant formula (MF) during the first 21 days of life. At postnatal day 21 (PND 21), a subset of piglets from each diet group (n = 11 per group) was euthanized. The remaining piglets in each group were weaned to a solid diet and euthanized at PND 51 (n = 13 per group). Large intestine contents (i.e., cecum and distal colon) were subjected to shotgun metagenomics analysis. The differential taxonomic composition of bacteria and fungi and the predicted functional gene profiling were evaluated. Bacteroidetes, Firmicutes, Proteobacteria, and Actinobacteria are the most abundant bacterial phyla observed in piglets at PND 21 and PND 51. In the large intestine at PND 21 and PND 51, Proteobacteria phylum was significantly higher in MF-fed group, and species Burkholderiales bacterium of phyla was significantly higher in MF group relative to HM group. In addition, in HM group, several Lactobacillus spp. and Bacteroides spp. were higher relative to MF group in the large intestine at PND 21 and PND 51. Fungal genus Aspergillus was higher in MF, whereas Malassezia was lower relative to HM group. Persistent effects of the neonatal diets were observed at PND 51, where alpha- and beta-diversity differences were detected for bacterial and fungal species in the large intestine. Overall, our findings indicate that neonatal diet affects the large intestinal microbial community during the exclusive milk-feeding period, as well as after the introduction of the complementary food.

In vivo transcriptional analysis of mice infected with Leishmania major unveils cellular heterogeneity and altered transcriptomic profiling at single-cell resolution.

Leishmania parasites cause cutaneous leishmaniasis (CL), a disease characterized by disfiguring, ulcerative skin lesions. Both parasite and host gene expression following infection with various Leishmania species has been investigated in vitro, but global transcriptional analysis following L. major infection in vivo is lacking. Thus, we conducted a comprehensive transcriptomic profiling study combining bulk RNA sequencing (RNA-Seq) and single-cell RNA sequencing (scRNA-Seq) to identify global changes in gene expression in vivo following L. major infection. Bulk RNA-Seq analysis revealed that host immune response pathways like the antigen processing and presentation pathway were significantly enriched amongst differentially expressed genes (DEGs) upon infection, while ribosomal pathways were significantly downregulated in infected mice compared to naive controls. scRNA-Seq analyses revealed cellular heterogeneity including distinct resident and recruited cell types in the skin following murine L. major infection. Within the individual immune cell types, several DEGs indicative of many interferon induced GTPases and antigen presentation molecules were significantly enhanced in the infected ears including macrophages, resident macrophages, and inflammatory monocytes. Ingenuity Pathway Analysis of scRNA-Seq data indicated the antigen presentation pathway was increased with infection, while EIF2 signaling is the top downregulated pathway followed by eIF4/p70S6k and mTOR signaling in multiple cell types including macrophages, blood and lymphatic endothelial cells. Altogether, this transcriptomic profile highlights known recruitment of myeloid cells to lesions and recognizes a potential role for EIF2 signaling in murine L. major infection in vivo.

In vivo ultrastructural analysis of the intimate relationship between polymorphonuclear leukocytes and the chlamydial developmental cycle.

We utilized a recently developed model of intracervical infection with Chlamydia muridarum in the mouse to elicit a relatively synchronous infection during the initial developmental cycle in order to examine at the ultrastructural level the development of both the chlamydial inclusion and the onset of the inflammatory response. At 18 h after infection, only a few elementary bodies attached to cells were visible, as were an occasional intracellular intermediate body and reticulate body. By 24 h, inclusions had 2 to 5 reticulate bodies and were beginning to fuse. A few polymorphonuclear leukocytes (PMNs) were already present in the epithelium in the vicinity of and directly adjacent to infected cells. By 30 h, the inclusions were larger and consisted solely of reticulate bodies, but by 36 to 42 h, they contained intermediate bodies and elementary bodies as well. Many PMNs were adjacent to or actually inside infected cells. Chlamydiae appeared to exit the cell either (i) through disintegration of the inclusion membrane and rupture of the cell, (ii) by dislodgement of the cell from the epithelium by PMNs, or (iii) by direct invasion of the infected cell by the PMNs. When PMNs were depleted, the number of released elementary bodies was significantly greater as determined both visually and by culture. Interestingly, depletion of PMNs revealed the presence of inclusions containing aberrant reticulate bodies, reminiscent of effects seen in vitro when chlamydiae are incubated with gamma interferon. In vivo evidence for the contact-dependent development hypothesis, a potential mechanism for triggering the conversion of reticulate bodies to elementary bodies, and for translocation of lipid droplets into the inclusion is also presented.

Essential role for neutrophils in pathogenesis and adaptive immunity in Chlamydia caviae ocular infections.

Trachoma, the world's leading cause of preventable blindness, is produced by chronic ocular infection with Chlamydia trachomatis, an obligate intracellular bacterium. While many studies have focused on immune mechanisms for trachoma during chronic stages of infection, less research has targeted immune mechanisms in primary ocular infections, events that could impact chronic responses. The goal of this study was to investigate the function of neutrophils during primary chlamydial ocular infection by using the guinea pig model of Chlamydia caviae inclusion conjunctivitis. We hypothesized that neutrophils help modulate the adaptive response and promote host tissue damage. To test these hypotheses, guinea pigs with primary C. caviae ocular infections were depleted of neutrophils by using rabbit antineutrophil antiserum, and immune responses and immunopathology were evaluated during the first 7 days of infection. Results showed that neutrophil depletion dramatically decreased ocular pathology, both clinically and histologically. The adaptive response was also altered, with increased C. caviae-specific IgA titers in tears and serum and decreased numbers of CD4(+) and CD8(+) T cells in infected conjunctivae. Additionally, there were changes in conjunctival chemokines and cytokines, such as increased expression of IgA-promoting interleukin-5 and anti-inflammatory transforming growth factor ß, along with decreased expression of T cell-recruiting CCL5 (RANTES). This study, the first to investigate the role of neutrophils in primary chlamydial ocular infection, indicates a previously unappreciated role for neutrophils in modulating the adaptive response and suggests a prominent role for neutrophils in chlamydia-associated ocular pathology.

Milk Formula Diet Alters Bacterial and Host Protein Profile in Comparison to Human Milk Diet in Neonatal Piglet Model.

The metaproteome profiling of cecal contents collected from neonatal piglets fed pasteurized human milk (HM) or a dairy-based infant formula (MF) from postnatal day (PND) 2 to 21 were assessed. At PND 21, a subset of piglets from each group (n = 11/group) were euthanized, and cecal contents were collected for further metaproteome analysis. Cecal microbiota composition showed predominantly more Firmicutes phyla and Lachnospiraceae family in the lumen of cecum of HM-fed piglets in comparison to the MF-fed group. Ruminococcus gnavus was the most abundant species from the Firmicutes phyla in the cecal contents of the HM-fed piglets at 21 days of age. A greater number of expressed proteins were identified in the cecal contents of the HM-fed piglets relative to the MF-fed piglets. Greater abundances of proteins potentially expressed by Bacteroides spp. such as glycoside enzymes were noted in the cecal lumen of HM-fed piglets relative to the MF. Additionally, lyases associated with Lachnospiraceae family were abundant in the cecum of the HM group relative to the MF group. Overall, our findings indicate that neonatal diet impacts the gut bacterial taxa and microbial proteins prior to weaning. The metaproteomics data were deposited into PRIDE, PXD025432 and 10.6019/PXD025432.