Therapeutic compounds and methods

Abstract

The invention provides methods for modulating intestinal immune function, modulating inflammatory responses, treating an inflammatory disorder, treating an autoimmune disorder, treating an allergic disorder, or treating a degenerative disorder in an animal by modulating the amount of one or more phosphatidylglycerol compounds in the animal (e.g., by oral administration of a phosphatidylglycerol compound).

Description

[0001] THERAPEUTIC COMPOUNDS AND METHODS

[0002] CROSS-REFERENCE TO RELATED APPLICATION

[0003] This application claims priority to United States Provisional Application Number 63 / 687,132 that was filed on August 26, 2024. The entire content of the application referenced above is hereby incorporated by reference herein.

[0004] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under AI122285 awarded by National Institutes of Health. The government has certain rights in the invention.

[0006] BACKGROUND

[0007] Using dietary substrates and products of host metabolism, gut microbes synthesize a vast array of small molecules (Tarracchini, C. et al., Front Microbiol 13, 1006721, (2022); Bishai, J. D. & Palm, N. W ., J Immunol 207, 1725-1733, (2021); Schoeler, M. et al., Nat Commun 14, 5329, Mucosal Immunol 15, 1095-1113 (2022); and Hitch, T. C. A. et al., Mucosal Immunol 15, 1095-1113, (2022)). These in turn significantly influence host metabolism, regulating the immune and nervous systems, impacting host physiology, and affect health and disease (Bishai, J. D. & Palm, N. W ., J Immunol 207 , 1725-1733, (2021); Luber, J. M. & Kostic, A. D., Curr Biol 27, R307-R310, (2017); Koundouros, N. & Poulogiannis, G.. Br J Cancer 122, 4-22, (2020); Dekkers, K. F. et al., Nat Commun 13, 5370, (2022); and Chaudhari, S. N., McCurry, M. D. & Devlin, A. S., Nat Chem Biol 17, 1046-1056, (2021)). Early life is the critical period for establishing the gut microbiome and facilitating healthy development of metabolism, immune and nervous systems (Huang, H., et al., EClinicalMedicine 68, 102428, (2024); Wemroth, M. L. et al., Sci Rep 12, 9080, (2022); and Hoskinson, C. et al., Nat Commun 14, 4785, (2023)). Environmental factors, including antibiotic exposures, altering gut microbiota composition can lead to abnormal development, increasing risk of subsequent metabolic and immune-mediated diseases (Colombo, J., Gustafson, K. M. & Carlson, S. E., Ann Nutr Metab 75 Suppl 1, 34-42, (2019); Pollak, S. D., Dev Psychopathol 17, 735-752, (2005); and Gollwitzer, E. S. & Marsland, B. J., Trends Immunol 36, 684-696, (2015)).

[0008] Type 1 diabetes (T1D), an autoimmune disease usually beginning in childhood, is characterized by the destruction of pancreatic insulin-producing beta cells by immune effectors (Eisenbarth, G. S., N Engl JMed 314, 1360-1368, (1986); and Atkinson, M. A. & Eisenbarth, G. S., Lancet 358, 221-229, (2001)). The gut microbiota with its effects on host immunity may play an important role in the early stages of T1D pathogenesis (Pino, S. C., Kruger, A. J. & Bortell, R., Curr Opin Endocrinol Diabetes Obes 17, 126-130, (2010); Del Chierico, F. et al., Int J Mol Set 23, (2022); and Zheng, P., Li, Z. & Zhou, Z . Diabetes Metab Res Rev 34, e3043, (2018)). Recent studies using the non-obese diabetic (NOD) mouse model revealed that early-life antibiotic exposure perturbs the gut microbiota, interferes with innate and adaptive immune effectors, alters ileal gene developmental patterns, and increases the incidence of T1D onset (Livanos, A. E. et al., Nat Microbiol 1, 16140, (2016); Zhang, X. S. et al., Elife 7, (2018); and Zhang, X. S. et al., Cell Host Microbe 29, 1249-1265 el249, (2021)). Maternal cecal microbiota transplant (CMT) to NOD mice after early-life antibiotic exposure significantly mitigated the induced T1D enhancement by partially restoring microbial diversity, relative abundance of specific taxa, and depleted metabolic pathways (Zhang, X. S. et al., Cell Host Microbe 29, 1249-1265 el249, (2021)); metagenomic analysis highlighted bacterial fatty acid metabolic pathways as differential between the experimental groups (Zhang, X. S. et al., Cell Host Microbe 29, 1249-1265 el249, (2021)).

[0009] Gut microbiota members produce diverse lipid molecules, including metabolites such as short-chain fatty acids which affect host metabolism, and bacterial membrane lipid molecules directly sensed by the host and modulating innate and adaptive immune pathways (Ghazalpour, A., Cespedes, I., Bennett, B. J. & Allayee, H., Curr Opin Lipidol 27 , 141-147, (2016); Brown, E. M., Clardy, J. & Xavier, R. J., Cell Host Microbe 31, 173-186, (2023); and Shiratori, H. et al., Sci Rep 13, 8903, (2023)). Although these may influence progression of chronic inflammation, allergic responses, and autoimmune diseases (Shiratori, H. et al., Sci Rep 13, 8903, (2023); Schoeler, M. & Caesar, R., Rev Endocr Metab Disord 20, 461-472, (2019); An,

[0010] D. et al., T cells. Cell 156, 123-133, (2014); and Lin, D. L. et al., J Immunol Res 2024, 2506586, (2024)), including T1D, interactions of hosts with intrinsic bacterial lipids have been understudied, with the notable exception of lipopolysaccharides (LPS) (Teng, O., Ang, C. K. E. & Guan, X. L., Front Immunol 8, 1836, (2017); and Chandler, C. E. & Ernst, R. K., FlOOORes 6, (2017)). Bacterially-derived lipids also include highly diverse and abundant phospholipids, released from bacterial cell membranes (Shiratori, H. et al., Sci Rep 13, 8903, (2023); Schoeler, M. & Caesar, R., Rev Endocr Metab Disord 20, 461-472, (2019); and Ryan,

[0011] E., Joyce, S. A. & Clarke, D. J., Microbiology (Reading) 169, (2023)). Phospholipids vary in the number and length of acyl-chains, the degree of unsaturation, whether iso-branching, and in head group composition such as choline, glycerol, inositol, and ethanolamine to produce lysoPC or PC, lysoPG or PG, lysoPI or PI, and lysoPE or PE, respectively (van Meer, G., Voelker, D. R. & Feigenson, G. W ., Nat Rev Mol Cell Biol 9 , 112-124, (2008); Sohlenkamp, C. & Geiger, O., FEMS Microbiol Rev 40, 133-159, (2016); and Strahl, H. & Errington, J., Annu Rev Microbiol 71, 519-538, (2017)). Such variation not only reflects the biochemical and biophysical characteristics of bacterial membranes enabling bacterial adaptation in the complex gut environment, but also could differentially mediate host-microbe communication modifying host immune responses (Tan, S. T., Ramesh, T., Toh, X. R. & Nguyen, L. N., Prog Lipid Res 80, 101068, (2020); and Ryan, E., Joyce, S. A. & Clarke, D. J., Microbiology (Reading) 169, (2023); and Tan, S. T., Ramesh, T., Toh, X. R. & Nguyen, L. N., Prog Lipid Res 80, 101068, (2020)).

[0012] Currently there is a need for compositions and methods that are useful for modulating intestinal immune function, modulating inflammatory response, treating inflammation, treating an autoimmune disorder, or treating a degenerative disease (e.g., a neurodegenerative diseases).

[0013] SUMMARY

[0014] In vitro co-culture experiments involving mouse macrophage, human colonic epithelial cells, and mouse small intestinal epithelial cells showed that phospholipids LPG(13:0), LPG(16:0), LPG(18:0) and PG(15:0_15:0) repress host inflammation through the NFKB pathway. Administering these phospholipids in early age to antibiotic-exposed NOD mice reduced specific ileal and colonic early gene expression markers of T1D pathogenesis. These findings suggest potential therapeutic roles of specific microbially-produced lipid compounds in mitigating risk for T1D and other inflammatory disorders.

[0015] A method comprising, modulating intestinal immune function in an animal, or modulating inflammatory responses in the gut of an animal, by modulating the amount of one or more phosphatidylglycerol compounds in the animal is provided.

[0016] A method to treat an inflammatory disorder, autoimmune disorder, an allergic disorder, and / or a degenerative disorder in an animal, comprising modulating the amount of one or more phosphatidylglycerol compounds in the animal is provided.

[0017] A method to restore integrity of gut epithelium in an animal following antibiotic treatment, comprising modulating the amount of one or more phosphatidylglycerol compounds in the animal is provided.

[0018] A method to improve mitochondrial function in epithelial cells (e.g., improve resilience and / or metabolic function) in an animal comprising modulating the amount of one or more phosphatidylglycerol compounds in the animal is provided.

[0019] A method to restore gene expression in an animal in which gene expression has been altered, comprising modulating the amount of one or more phosphatidylglycerol compounds in the animal is provided. In some embodiments, the gene expression has been altered by an inflammatory process, antibiotic treatment, or cancer.

[0020] A method to restore gene expression in an animal in which gene expression of the gut epithelium (e.g. ileal) has been altered (e.g., deranged) by antibiotic treatment, comprising modulating the amount of one or more phosphatidylglycerol compounds in the animal is provided.

[0021] A method to mitigate tissue injury in an animal, comprising modulating the amount of one or more phosphatidylglycerol compounds in the animal is provided. In some embodiments, the tissue injury is associated with an inflammatory process, antibiotic treatment, or cancer.

[0022] A method to restore the level of a phosphatidylglycerol compound in an animal, comprising administering the phosphatidylglycerol compound to the animal is provided. In some embodiments, the level of the phosphatidylglycerol compound has been reduced by an inflammatory process, antibiotic treatment, or cancer.

[0023] A method to restore a level of a phosphatidylglycerol compound in an animal, comprising administering the phosphatidylglycerol compound to the animal is provided. In some embodiments, a unit dosage form that comprises the phosphatidylglycerol compound is administered to the animal.

[0024] A pharmaceutical composition comprising a phosphatidylglycerol compound and a pharmaceutically acceptable excipient is provided. In some embodiments, the pharmaceutical composition is formulated as a unit dosage form.

[0025] A pharmaceutical composition comprising a phosphatidylglycerol compound for modulating intestinal immune function in an animal, modulating an inflammatory response in the gut of an animal, treating an inflammatory disorder, treating autoimmune disorder, treating an allergic disorder, and / or treating a degenerative disorder in an animal is provided. In some embodiments, the pharmaceutical composition is formulated as a unit dosage form.

[0026] A phosphatidylglycerol compound for use in medical therapy is provided. In some embodiments, the phosphatidylglycerol compound is formulated in a unit dosage form.

[0027] A phosphatidylglycerol compound for modulating intestinal immune function or modulating inflammatory response is provided. In some embodiments, the phosphatidylglycerol compound is formulated in a unit dosage form.

[0028] A phosphatidylglycerol compound for the prophylactic or therapeutic treatment of an inflammatory disorder, an autoimmune disorder, an allergic disorder, or a degenerative disorder is provided. In some embodiments, the phosphatidylglycerol compound is formulated in a unit dosage form.

[0029] A phosphatidylglycerol compound for restoring integrity of gut epithelium in an animal following antibiotic treatment is provided. In some embodiments, the phosphatidylglycerol compound is formulated in a unit dosage form. A phosphatidylglycerol compound for improving mitochondrial function in epithelial cells is provided. In some embodiments, the phosphatidylglycerol compound is formulated in a unit dosage form.

[0030] A phosphatidylglycerol compound for restoring gene expression in an animal in which gene expression has been altered is provided. In some embodiments, the gene expression has been altered by an inflammatory process, antibiotic treatment, or cancer. In some embodiments, the phosphatidylglycerol compound is formulated in a unit dosage form.

[0031] A phosphatidylglycerol compound for restoring gene expression of the gut epithelium (e.g., ileal) in an animal in which gene expression of the gut epithelium (e.g., ileal) has been altered (e.g., deranged) by antibiotic treatments provided. In some embodiments, the phosphatidylglycerol compound is formulated in a unit dosage form.

[0032] A phosphatidylglycerol compound for mitigating tissue injury in an animal. In some embodiments, the tissue injury is associated with an inflammatory process, antibiotic treatment, or cancer. In some embodiments, the phosphatidylglycerol compound is formulated in a unit dosage form.

[0033] A phosphatidylglycerol compound for restoring a level of a phosphatidylglycerol compound in an animal is provided. In some embodiments, the phosphatidylglycerol compound is formulated in a unit dosage form.

[0034] Use of a phosphatidylglycerol compound to prepare a medicament for modulating intestinal immune function or modulating inflammatory response in an animal is provided. In some embodiments, the medicament is formulated in a unit dosage form.

[0035] Use of a phosphatidylglycerol compound to prepare a medicament for treating an inflammatory disorder, treating autoimmune disorder, treating an allergic disorder, or treating a degenerative disorder in an animal is provided. In some embodiments, the medicament is formulated in a unit dosage form.

[0036] Use of a phosphatidylglycerol compound to prepare a medicament for restoring integrity of gut epithelium in an animal following antibiotic treatment is provided. In some embodiments, the medicament is formulated in a unit dosage form.

[0037] Use of a phosphatidylglycerol compound to prepare a medicament for improving mitochondrial function in epithelial cells in an animal is provided. In some embodiments, the medicament is formulated in a unit dosage form.

[0038] Use of a phosphatidylglycerol compound to prepare a medicament for restoring gene expression in an animal in which gene expression has been altered is provided. In some embodiments, the gene expression has been altered by an inflammatory process, antibiotic treatment, or cancer. In some embodiments, the medicament is formulated in a unit dosage form.

[0039] Use of a phosphatidylglycerol compound to prepare a medicament for restoring gene expression of the gut epithelium (e.g., ileal) in an animal in which gene expression of the gut epithelium (e.g., ileal) has been altered (e.g., deranged) by antibiotic treatment is provided. In some embodiments, the medicament is formulated in a unit dosage form.

[0040] Use of a phosphatidylglycerol compound to prepare a medicament for mitigating tissue injury in an animal is provided. In some embodiments, the gene expression has been altered by an inflammatory process, antibiotic treatment, or cancer. In some embodiments, the medicament is formulated in a unit dosage form.

[0041] Use of a phosphatidylglycerol compound to prepare a medicament for restoring a level of the phosphatidylglycerol compound in an animal. In some embodiments, the medicament is formulated in a unit dosage form.

[0042] BRIEF DESCRIPTION OF DRAWINGS

[0043] Figs. 1A-1D show the repressive effects of LPG(13:0), PG(15:0_15:0), LPG(16:0), and LPG(18:0) on LPS-induced NFKB activity in mouse macrophage Raw Blue cells, in panels A, B, C, D, respectively. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001; Mann-Whitney U-test.

[0044] Figs. 2A-2D show effects of LPG(13:0), PG(15:0_15:0), LPG(16:0), and LPG(18:0) on the expression of particular innate immune genes in human colonic epithelial HT-29 cells during co-culturing, in panels A, B, C, D, respectively. *p<0.05; **p<0.01; Mann-Whitney U-test.

[0045] Figs. 3A-3B show effects of LPG(13:0) and PG(15:0_15:0) on the expression of particular innate immune genes in primary small intestinal epithelial cells (SIECs) from male NOD mice at P23, in panels A and B, respectively. *p<0.05; **p<0.01; Mann-Whitney U-test.

[0046] Fig- 4 shows study design and intestinal gene expression analysis of NOD mice exposed to early-life antibiotics and then administered defined lipid compounds for restoration. Pregnant NOD / ShiLtJ mice were randomized into 8 groups. Pups in 7 groups received a tylosin course (1PAT; IP) during days P5-P10. Then from P12-P18, five of the IP groups were each gavaged daily with one of five defined lipid compounds, LPG(13:0), PG(15:0_15:0), LPG(16:0), LPG(18:0), or retinoic acid (RA), or with cecal material transplant from male P23 NOD pups, or with blank solvent (PBS-2% EtOH) only. One group without a tylosin course (Control) was gavaged with the blank solvent from P12-P18 as control. For each group, four pups were sacrificed atP19 to assess effects of the administered lipid compound on intestinal gene expression. Fig. 5 shows results from Example 4. RT-qPCR-based Pl 9 ileal gene expression of REG3y, NOS2, and IL12. Mean±SD of loglO values of relative expression shown. *p<0.05 compared with IP group; Mann-Whitney V-test.

[0047] Fig. 6 shows the structures for LPG(13:0), PG(15:0 15:0), LPG(16:0), and LPG(18:0).

[0048] Fig- 7 shows the key enzymes in the gut bacterial pathway of phosphatidylglycerol (PG) and lyso-phosphatidylglycerol (LPG) biosynthesis, with the relative abundance of genes altered by antibiotic exposure (IP) and restored by cecal material transplant (CMT), compared with control (C), based on metagenomic analysis of the cecal microbiome in NOD mice at P23.

[0049] *p<0.05; **p<0.01; ***p<0.001 by Mann-Whitney { / -test.

[0050] Figs. 8A-8J. show the effects of LPG16:0 and LPG18:0 on mitochondrial respiration and ATP production in HT29 intestinal epithelial cells. HT29 cells were treated with increasing concentrations of LPG16:0 (0.5, 2.0, and 5.0 pM) or LPG18:0 (0.5, 1.0, and 2.0 pM), or blank reagent (PBS) only in DMEM medium with 10 mM glucose for 24 h, and mitochondrial respiration was assessed using the Seahorse XF Analyzer. Time-course plots of oxygen consumption rate (OCR) following sequential injection of oligomycin, FCCP, and rotenone / antimycin A in cells treated with LPG16:0 (A) or LPG18:0 (F). Data represent mean ± SEM. In panels A and F, the PBS line is the bottom dark line, and the lines above show the effects of the LPGs in order by concentration. Quantitation of basal respiration (B), maximal respiration (C), ATP-linked respiration (D), and non-mitochondrial respiration (E) in response to LPG16:0 treatment. Corresponding measurements for LPG18:0 treatment: basal respiration (G), maximal respiration (H), ATP-linked respiration (I), and non-mitochondrial respiration (J). Data represent mean ± SD. Statistical significance determined by Mann-Whitney U-test. *p<0.05, **p<0.01.

[0051] Figs 9A-9D show the effects in vivo of LPG16:0 and LPG18:0 administration after antibiotic treatment. Experiments performed in which control NOD mice were compared with those that received an early-life antibiotic course (IP) and then one of the specified phospholipids or not (n=5 mice / group). Principal coordinate analysis (PCoA) of ileal transcriptomes based on variance-stabilizing transformation (VST)-normalized counts for 21,924 transcripts. Statistical significance determined using adjusted p < 0.05. (A) Significantly differentiated KEGG pathways after administration of LPG16:0 compared to 1PAT after antibiotic-exposure, indicates two pathways (phagosome and proteasome) that were downregulated by LPG16:0 in 1PAT mice and are closely linked to mitochondrial physiology and functions. (B) Volcano plots of differential genes in pairwise comparisons of groups: 1PAT vs Control (Left; n=307 significantly different), LPG16:0 vs Control (Middle; n=36 significantly different), and LPG18:0 vs Control (Right; n=49 significantly different). (C) Venn diagram of comparisons with Control of differentially expressed genes (DEGs) in antibiotic-treated (1PAT) NOD mice following buffer alone (1PAT; n=307) or administration of LPG16:0 (n=36), or LPG18:0 (n=49). Numbers indicate total DEGs in each of Regions I- VII, with arrows indicating number of upregulated ) and downregulated (J,) genes, respectively. The relatively small number of DEGs in the LPG16:0 and LPG18:0 groups indicates that both phospholipid treatments largely restored gene expression toward baseline (control) levels. (D) From the unsupervised hierarchical clustering of the 270 genes in which IP was significantly differentiated from control and restored by LPG16:0 and LPG18:0 (Region I in 8C), KEGG pathway enrichment analysis [of these 270 Region I genes] identifying pathways disrupted by IP and subsequently restored toward control levels by both LPG16:0 and LPG18:0.

[0052] Fig. 10 shows RNAseq analysis of ileal gene expression after LPG16:0 and LPG18:0 administration in antibiotic-treated NOD mice. Top: Significantly differentiated KEGG pathways comparing 1PAT alone or followed by administration ofLPG18:0. Bottom: Representative genes modulated by treatment. Trim40, Sprr2al, and Socsl were up-regulated by antibiotics (IP) but returned to baseline with either lipid, whereas the uncharacterized transcript A930038B10Rik was down-regulated by IP and no longer was significantly by both LPG16:0 and LPG18:0. Data represent mean ± SD. Statistical significance determined by Mann-Whitney U-test. *p<0.05, **p<0.01

[0053] DETAILED DESCRIPTION

[0054] The following definitions are used, unless otherwise described: halo or halogen is fluoro, chloro, bromo, or iodo. Alkyl, alkoxy, etc. denote both straight and branched groups; but reference to an individual radical such as propyl embraces only the straight chain radical, a branched chain isomer such as isopropyl being specifically referred to.

[0055] The term "alkyl", by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain hydrocarbon radical, having the number of carbon atoms designated (i.e., Ci-s means one to eight carbons). Examples include (Ci-Cs)alkyl, (C2-Cs)alkyl, Ci-Ce)alkyl, (C2-Ce)alkyl and (C3-Ce)alkyl. Examples of alkyl groups include methyl, ethyl, n- propyl, iso-propyl, n-butyl, t-butyl, iso-butyl, sec-butyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, and and higher homologs and isomers.

[0056] The term "alkoxy" refers to an alkyl groups attached to the remainder of the molecule via an oxygen atom (“oxy”).

[0057] The term “alkanoyloxy” as used herein refers to a group (alkyl)-C(=O)-O-, wherein the term alkyl has the meaning defined herein.

[0058] As used herein, the term "heteroatom" is meant to include oxygen (O), nitrogen (N), sulfur (S) and silicon (Si). As used herein, the term "protecting group" refers to a substituent that is commonly employed to block or protect a particular functional group on a compound. For example, an "amino-protecting group" is a substituent attached to an amino group that blocks or protects the amino functionality in the compound. Suitable amino-protecting groups include acetyl, trifluoroacetyl, t-butoxycarbonyl (BOC), benzyloxycarbonyl (CBZ) and 9- fluorenylmethylenoxycarbonyl (Fmoc). Similarly, a "hydroxy-protecting group" refers to a substituent of a hydroxy group that blocks or protects the hydroxy functionality. Suitable protecting groups include acetyl and silyl. A "carboxy-protecting group" refers to a substituent of the carboxy group that blocks or protects the carboxy functionality. Common carboxy- protecting groups include phenylsulfonylethyl, cyanoethyl, 2-(trimethylsilyl)ethyl, 2- (trimethylsilyl)ethoxymethyl, 2-(p-toluenesulfonyl)ethyl, 2-(p-nitrophenylsulfenyl)ethyl, 2- (diphenylphosphino)-ethyl, nitroethyl and the like. For a general description of protecting groups and their use, see P.G.M. Wuts and T.W. Greene, Greene's Protective Groups in Organic Synthesis 4thedition, Wiley-Interscience, New York, 2006.

[0059] As used herein, the term “inflammatory disorder” includes, but is not limited to inflammatory bowel disease (including Crohn’s disease and ulcerative colitis), gout, and psoriasis.

[0060] As used herein, the term “autoimmune disorder” includes, but is not limited to type 1 diabetes, Celiac disease, multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus (SLE) and pemphigus.

[0061] As used herein, the term “allergic disorder” includes, but is not limited to asthma (allergic type), allergic rhinitis (hay fever), food allergies, and drug allergies.

[0062] As used herein, the term “degenerative disorder” includes, but is not limited to Alzheimer’s disease, Parkinson’s disease, and atherosclerotic cardiovascular diseases (ASCVD).

[0063] For each of the terms “treat”, “treatment”, or “treating” to the extent that it relates to a disease or condition includes inhibiting the disease or condition, eliminating the disease or condition, and / or relieving one or more symptoms of the disease or condition. The terms “treat”, “treatment”, or “treating” also refer to both therapeutic treatment and / or prophylactic treatment or preventative measures, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as, for example, the development or spread of an inflammatory, auto-immune or degenerative disease, or cancer. For example, beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease or disorder, stabilized (i.e., not worsening) state of disease or disorder, delay or slowing of disease progression, amelioration or palliation of the disease state or disorder, and remission (whether partial or total), whether detectable or undetectable. “Treat”, “treatment”, or “treating,” can also mean prolonging survival as compared to expected survival if not receiving treatment. Those in need of treatment include those already with the disease or disorder as well as those prone to have the disease or disorder or those in which the disease or disorder is to be prevented. In some embodiments “treat”, “treatment”, or “treating” does not include preventing or prevention,

[0064] The phrase "therapeutically effective amount" or “effective amount” includes but is not limited to an amount of a compound of the that (i) treats or prevents the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein.

[0065] The term “animal” includes mammals, fish, amphibians, reptiles, birds and invertebrates. The term “mammal” includes humans, higher non-human primates, rodents, domestic, cows, horses, pigs, sheep, dogs and cats. In some embodiments, the animal is a mammal. In some embodiments, the animal is a human. The term “patient” as used herein refers to any animal including mammals. In some embodiments, the patient is a mammalian patient. In some embodiments, the patient is a human patient.

[0066] In some embodiments, the phosphatidylglycerol compound can be administered to the animal as part of the animal’s diet. In some embodiments, the phosphatidylglycerol compound can be added to the animal’s diet, so that it is present in an amount greater than the amount present in a normal diet, e.g., the animal’s food is supplemented with the phosphatidylglycerol compound.

[0067] As used herein, the term “unit dose” means the amount of an active agent administered to a patient in a single dose. As used herein, the term “unit dosage form” includes dosage forms (e.g., tablets, capsules, single use vials, etc.) that are formulated to include the amount of an active agent to be administered to a patient in a single dose. In some embodiments, the term unit dosage form excludes bulk liquids administered to an animal as well as the animal’s unsupplemented diet.

[0068] Modulating the amount of one or more phosphatidylglycerol compounds

[0069] As used herein, the term, “modulate the amount of one or more phosphatidylglycerol compounds in an animal” includes any action that results in a change (e.g., an increase) in the amount of one or more phosphatidylglycerol compounds in an animal. For example, the amount of one or more phosphatidylglycerol compounds in an animal can be increased by delivering a pre-biotic that increases the presence of one or more bacterial strains that produce the one or more phosphatidylglycerol compounds in the animal. The amount of one or more phosphatidylglycerol compounds in an animal can also be increased by inhibiting one or more bacterial enzymes that degrade phosphatidylglycerol compound(s) in the animal. The amount of one or more phosphatidylglycerol compounds in an animal can also be increased by stimulating bacterial enzymes that are involved, directly or indirectly, in the synthesis of the phosphatidylglycerol compound(s). The amount of one or more phosphatidylglycerol compounds in an animal can also be increased by administering the one or more phosphatidylglycerol compound(s) or prodrugs thereof to the animal.

[0070] Figure 7 shows the key enzymes in the gut microbial pathway of phosphatidylglycerol (PG) and lysyl-phosphatidylglycerol (LPG) biosynthesis with the relative abundance of the relevant genes that were identified in the NOD mouse model at day of life 23 (P23). The key enzymes include: (i),FabH (P-ketoacyl-ACP synthase III) (encoded by fabH), which plays a critical role in initiating fatty acid synthesis and regulates the formation of odd-chain and evenchain PG and LPG fatty acid structures; (ii), PgsA (phosphatidylglycerophosphate synthase), encoded by pgsA, which controls the crucial step in bacterial phospholipid biosynthesis, converting cytidine diphosphate-diacylglycerol (CDP-DAG) and glycerol -3 -phosphate (G3P) into phosphatidylglycerophosphate (PGP); (iii), PgpA, PgpB, and PgpC (phosphatidylglycerolphosphatases PgpA, B, C), encoded by pgpA, pgpB, pgpC, respectively, are involved in the final step of bacterial PG biosynthesis from PGP (The functional redundancy of the 3 genes, all contributing to the production of PG help bacteria maintain a steady supply of PG, crucial for membrane integrity and function); (iv) PLA2 (phospholipase A2 ) plays a critical role in the metabolism of phospholipids by hydrolyzing the sn-2 acyl glycerophospholipid bond to generate lysophospholipids; and (v) ClsA, ClsB, and ClsC (Cardiolipin Synthases A, B, C), encoded by clsA, clsB, clsC, respectively, convert PG into cardiolipin (CL). In the NOD mouse model, the abundances of the genes encoding these enzymes in the cecal microbiome were altered by early- life antibiotic exposure and subsequently restored by cecal material transplant. This suggests that modulating these key enzymes would enable stimulating further bacterial production of PG and LPG in the intestinal tract of treated animals.

[0071] Phosphatidylglycerol Compounds

[0072] The term “phosphatidylglycerol compound” includes compounds and salts that comprise a phosphate group and one or two hydrophobic "tails." In some embodiments, the phosphate group and the one or two hydrophobic tails can be joined by an alcohol residue (e.g., a residue of a glycerol molecule). In some embodiments, the phosphatidylglycerol compound is a compound of formula (I): or a salt thereof, wherein:

[0073] R1is a saturated or unsaturated, branched or straight hydrocarbon chain comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, which is optionally substituted with one or more halo groups;

[0074] R2is H or a saturated or unsaturated, branched or straight hydrocarbon chain comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, which is optionally substituted with one or more halo groups;

[0075] R3is OH, (Ci-Ce)alkoxy, (Ci-C6)cycloalkyloxy, or (Ci-Ce)alkanoyloxy, which (Ci- Ce)alkoxy, (Ci-C6)cycloalkyloxy, and (Ci-Ce)alkanoyloxy is optionally substituted with one or more groups independently selected from the group consisting of halo hydroxy, -N+(Ra)(Rb)(Rc)X-, and (Ci-C3)alkoxy;

[0076] Rais (Ci-C6)alkyl;

[0077] Rbis (Ci-C6)alkyl;

[0078] Rcis (Ci-Ce)alkyl; and

[0079] X is a suitable counter-anion.

[0080] In some embodiments, R1is a saturated hydrocarbon chain comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.

[0081] In some embodiments, R1is an unsaturated hydrocarbon chain comprising 12, 13, 14, 15,

[0082] 16, 17, 18, 19, or 20 carbon atoms.

[0083] In some embodiments, R1comprises one or two double bonds.

[0084] In some embodiments, R1comprises one double bond.

[0085] In some embodiments, R1comprises one or two triple bonds.

[0086] In some embodiments, R1comprises one triple bond.

[0087] In some embodiments, R1is a branched hydrocarbon chain.

[0088] In some embodiments, R1is a straight hydrocarbon chain.

[0089] In some embodiments, R1comprises 13, 15, 17, or 19 carbon atoms.

[0090] In some embodiments, R1comprises 12, 14, 16, 18, or 20 carbon atoms.

[0091] In some embodiments, R2is a saturated hydrocarbon chain comprising 12, 13, 14, 15, 16,

[0092] 17, 18, 19, or 20 carbon atoms.

[0093] In some embodiments, R2is an unsaturated hydrocarbon chain comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms. In some embodiments, R2comprises one or two double bonds.

[0094] In some embodiments, R2comprises one double bond.

[0095] In some embodiments, R2comprises one or two triple bonds.

[0096] In some embodiments, R2comprises one triple bond.

[0097] In some embodiments, R2is a branched hydrocarbon chain.

[0098] In some embodiments, R2is a straight hydrocarbon chain.

[0099] In some embodiments, R2comprises 13, 15, 17, or 19 carbon atoms.

[0100] In some embodiments, R2comprises 12, 14, 16, 18, or 20 carbon atoms.

[0101] In some embodiments, R2is H.

[0102] In some embodiments, R1and R2are the same.

[0103] In some embodiments, R1and R2are different.

[0104] In some embodiments, R3is OH.

[0105] In some embodiments, R3is (Ci-Ce)alkoxy that is optionally substituted with one or more groups independently selected from the group consisting of halo hydroxy, and (Ci- C3)alkoxy.

[0106] In some embodiments, R3is (Ci-Ce)alkoxy that is optionally substituted with one or more groups independently selected from the group consisting of halo hydroxy, and (Ci- C3)alkoxy.

[0107] In some embodiments, R3is (Ci-Ce)alkoxy that is substituted with one or more hydroxy groups.

[0108] In some embodiments, R3is 2,3-dihydroxypropoxy.

[0109] In some embodiments, R3is (Ci-Ce)alkanoyloxy that is optionally substituted with one or more groups independently selected from the group consisting of halo hydroxy, and (Ci- C3)alkoxy.

[0110] In some embodiments, R3is (Ci-Ce)alkoxy that is optionally substituted with -N+(Ra)(Rb)(Rc)X'; wherein Rais methyl; Rbis methyl; and Rcis methyl.

[0111] In some embodiments, R3is 2,3-dihydroxypropoxy, 2, 3, 4, 5, 6-pentahydroxy- cyclohexyloxy, or 2-(trimethylammonium chloride)ethoxy.

[0112] In some embodiments, X is chloro or bromo.

[0113] In some embodiments, the phosphatidylglycerol compound is LPG(13:0), LPG(16:0), or PG(15:0_15:0) or a salt thereof.

[0114] In some embodiments, the animal is a human.

[0115] In some embodiments, the animal has received antibiotic treatment that has caused an unwanted effect, due to a perturbed microbiome - and the unwanted effect is reduced by modulating the amount of one or more phosphatidylglycerol compounds in the animal. In some embodiments, the unwanted effect leads to the exacerbation of an inflammatory disorder, an autoimmune disorder, an allergic disorder, and / or a degenerative disorder. In some embodiments, the animal is in need of treatment for the unwanted effect.

[0116] In some embodiments, the animal has a disturbed immune response in the gut associated with a disease selected from the group consisting of Type 1 diabetes, inflammatory bowel diseases (including, Crohn’s disease, Ulcerative colitis, and Celiac disease); asthma, eczema, food allergies, allergic rhinitis (hay fever), psoriasis, type 2 diabetes, obesity, atherosclerotic cardiovascular disease (ASCVD), Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis, and the disturbed immune response is reduced by modulating the amount of one or more phosphatidylglycerol compounds in the animal. In some embodiments, the animal is in need of treatment to reduce the disturbed immune response.

[0117] In some embodiments, the animal has received antibiotic treatment that has changed gene expression in the intestine of the animal and the gene expression is restored (partially or fully) by modulating the amount of one or more phosphatidylglycerol compounds in the animal. In some embodiments, the animal is in need of treatment to restore the intestinal gene expression.

[0118] In some embodiments, the animal has an inflammatory condition that has changed gene expression in the intestine and the gene expression is restored (partially or fully) by modulating the amount of one or more phosphatidylglycerol compounds in the animal. In some embodiments, the animal is in need of treatment to restore the gene expression.

[0119] In some embodiments, cancer has led to altered intestinal gene expression in the animal and the gene expression is restored (partially or fully) by modulating the amount of one or more phosphatidylglycerol compounds in the animal. In some embodiments, the animal is in need of treatment to restore the gene expression.

[0120] As used herein, “modulation” is increasing or decreasing to provide a desired effect. In some embodiments, modulation may be evaluated by examining expression of a particular gene of interest. In some embodiments, modulation is a change of at least 0.5 logic unit relative expression with reference to a standard housekeeping gene in the cells or tissues being studied. In some embodiments, modulation is a change of at least 1.0 logic unit relative expression (10- fold). In some embodiments, modulation is a change of at least 1.5 logic unit relative expression. In some embodiments, modulation is a change of at least 2.0 logic unit relative expression (100-fold). The modulation can be either direction depending on the gene in question and clinical illness. In some embodiments, modulation includes a decrease in expression of a pro-inflammatory gene (for example, see the genes listed in Example 2, Figures 2, 3 including RoR-gamma-t, caspase, TLR5, or IL-13). In some embodiments, modulation includes an increase in genes related to the regulation of the immune response of an animal (for example, the genes shown in Example 4, Figure 5, Reg3-gamma, NOS-2, or IL-12)

[0121] In some embodiments, “restoration” is defined as the opposite effect or returning to normal, e.g., restoration of at least 0.5 logic unit relative expression of deviation from normal.

[0122] The ability of a phosphatidylglycerol compound to: modulate intestinal immune function, modulate an inflammatory response, treat an inflammatory disorder, treat an autoimmune disorder, treat an allergic disorder, treat a degenerative disorder, restore integrity of gut epithelium, improve mitochondrial function in epithelial cells, restore gene expression, mitigate tissue injury, and / or restore the level of the phosphatidylglycerol compound in an animal can be evaluated using the methods described herein (for example, the methods described in the Examples herein) or using methods that are known in the field.

[0123] The compounds disclosed herein can also exist as tautomeric isomers in certain cases. Although only one delocalized resonance structure may be depicted, all such forms are contemplated.

[0124] It is understood by one skilled in the art that this invention also includes any compound claimed that may be enriched at any or all atoms above naturally occurring isotopic ratios with one or more isotopes such as, but not limited to, deuterium (2H or D). As a non-limiting example, a -CH3 group may be substituted with -CD3.

[0125] The pharmaceutical compositions can comprise one or more excipients. When used in combination with the pharmaceutical compositions the term “excipients” refers generally to an additional ingredient that is combined with the compound of formula (I) or the pharmaceutically acceptable salt thereof to provide a corresponding composition. For example, when used in combination with the pharmaceutical compositions the term “excipients” includes, but is not limited to: carriers, binders, disintegrating agents, lubricants, sweetening agents, flavoring agents, coatings, preservatives, and dyes.

[0126] Stereochemical definitions and conventions used herein generally follow S. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984) McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S., "Stereochemistry of Organic Compounds", John Wiley & Sons, Inc., New York, 1994. The compounds can contain asymmetric or chiral centers, and therefore exist in different stereoisomeric forms. It is intended that all stereoisomeric forms of the compounds, including but not limited to, diastereomers, enantiomers and atropisomers, as well as mixtures thereof such as racemic mixtures, form part of the present invention. Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L, or R and S, are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the sign of rotation of plane- polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these stereoisomers are identical except that they are mirror images of one another. A specific stereoisomer can also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric mixture. A 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which can occur where there has been no stereoselection or stereospecificity in a chemical reaction or process. The terms "racemic mixture" and "racemate" refer to an equimolar mixture of two enantiomeric species, devoid of optical activity.

[0127] It will be appreciated by those skilled in the art that compounds having a chiral center may exist in and be isolated in optically active and racemic forms. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, polymorphic, or stereoisomeric form, or mixtures thereof, of a compound, which possess the useful properties described herein, it being well known in the art how to prepare optically active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, or by chromatographic separation using a chiral stationary phase.

[0128] When a bond in a compound formula herein is drawn in a non- stereochemical manner (e.g. flat), the atom to which the bond is attached includes all stereochemical possibilities. When a bond in a compound formula herein is drawn in a defined stereochemical manner (e.g. bold, bold-wedge, dashed or dashed-wedge), it is to be understood that the atom to which the stereochemical bond is attached is enriched in the absolute stereoisomer depicted unless otherwise noted. In some embodiments, the compound may be at least 51% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 60% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 80% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 90% the absolute stereoisomer depicted. In another embodiment, the compound may be at least 95 the absolute stereoisomer depicted. In another embodiment, the compound may be at least 99% the absolute stereoisomer depicted.

[0129] The term “residue” as it applies to the residue of a compound refers to a compound that has been modified in any manner which results in the creation of an open valence wherein the site of the open valence. The open valence can be created by the removal of 1 or more atoms from the compound (e.g., removal of a single atom such as hydrogen or removal of more than one atom such as a group of atoms including but not limited to an amine, hydroxyl, methyl, amide (e.g., -C(=O)NH2) or acetyl group). The open valence can also be created by the chemical conversion of a first function group of the compound to a second functional group of the compound (e.g., reduction of a carbonyl group, replacement of a carbonyl group with an amine, ) followed by the removal of 1 or more atoms from the second functional group to create the open valence.

[0130] Specific values listed below for radicals, substituents, and ranges, are for illustration only; they do not exclude other defined values or other values within defined ranges for the radicals and substituents. It is to be understood that two or more values may be combined. It is also to be understood that the values listed herein below (or subsets thereof) can be excluded.

[0131] In cases where compounds are sufficiently basic or acidic, a salt of a compound of formula (I) can be useful as an intermediate for isolating or purifying a compound of formula (I). Additionally, administration of a compound of formula (I) as a pharmaceutically acceptable acid or base salt may be appropriate. Examples of pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiological acceptable anion, for example, tosylate, methanesulfonate, acetate, citrate, malonate, tartrate, succinate, benzoate, ascorbate, a- ketoglutarate, and a-glycerophosphate. Suitable inorganic salts may also be formed, including hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts.

[0132] Salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. Alkali metal (for example, sodium, potassium or lithium) or alkaline earth metal (for example calcium) salts of carboxylic acids can also be made.

[0133] The compounds of formula (I) can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient in a variety of forms adapted to the chosen route of administration, i.e., orally or parenterally, by intravenous, intramuscular, topical or subcutaneous routes.

[0134] Thus, the present compounds may be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard-shell or soft-shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices.

[0135] The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

[0136] The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

[0137] Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.

[0138] For topical administration, the present compounds may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid.

[0139] Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include water, alcohols or glycols or water-alcohol / glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

[0140] Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user.

[0141] Examples of useful dermatological compositions which can be used to deliver the compounds of formula (I) to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508).

[0142] Useful dosages of the compounds of formula (I) can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949.

[0143] The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye.

[0144] The invention will now be illustrated by the following non-limiting Examples.

[0145] EXAMPLES

[0146] Example 1. Administration of phosphatidylglycerol compounds reduces sensitivity of inflammatory cells to endotoxin (LPS) stress. (Figure 1)

[0147] Materials and Methods

[0148] Lipid compound standards. SPLASH LipidoMIX Mass Spec Standard, including PC(15:0_18:l-d7), PE(15:0_18:l-d7), PS(15:0_18: l-d7), PG(15:0_18:l-d7), PI(15:0_18:l-d7), PA(15 :0_l 8 : 1 -d7), LPC(18 : 1 -d7), LPE(18 : 1 -d7), cholesterol(l 8 : 1 -d7), MG(18 : 1 -d7), DG(15:0_18: l-d7), TG(15:0_18: l-d7_15:0), SM(18: l-d7), and cholesterol(d7)] were purchased from Avanti Polar Lipids (cat# 330707, Birmingham, AL) and used as internal standards for lipidomic MS analysis. For use as references for specific cecal lipid MS2 analyses and for both in vitro and in vivo experiments, we purchased Lysophosphatidylglycerols LPG (13:0) [1- tridecanoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt)], LPG (16:0) [ 1 -palmitoyl -2- hydroxy-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt)], & LPG (18:0) [l-stearoyl-2- hydroxy-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt)], and phosphatidylglycerol PG (15:0 15:0) [l,2-dipentadecanoyl-sn-glycero-3-phospho-(l'-rac-glycerol) (sodium salt)] (Avanti Polar Lipids), and retinoic acid (RA) and E. coli lipopolysaccharides (LPS) was purchased from Sigma-Aldrich (cat# L2012-10MG, Saint Louis MO).

[0149] Preparation of lipids. Total lipids were extracted using the methyl tert-butyl ether (MTBE) method (Matyash, V., et al., J Lipid Res 49, 1137-1146 (2008)). Fresh extraction solvent was prepared on the same day of total lipid extraction by mixing 0.1 M hydrochloric acid (Sigma- Aldrich cat# 1090601000), methanol (Sigma-Aldrich cat#34860), and the SPLASH LipidoMIX Internal Standard in a 500:495:5 N / N / N ratio. For each 10-15 mg sample of cecal contents, feces, or mouse diet, 50 pL fresh extraction solvent was added to 2 mL screw cap tubes containing 200 pL of 0.1 mm glass beads (BioSpec cat# 11079101, Bartlesville OK). The samples were homogenized at 2000 rpm for 45 seconds, then paused for 45 seconds, repeating for 5 cycles to avoid overheating. The mixture was then combined with 2 volumes of MTBE, vortexed for 30 seconds, and left to stand at room temperature for 1 minute to allow layer separation. The top MTBE layer containing total lipids (600 pL) from each sample was collected and air-dried in a fume hood for ~16 hours. The dried samples were stored at -80°C until mass spectrometry analysis performed at the Metabolomics Shared Resource.

[0150] Cell culture and co-culture assays. Murine macrophage NFKB-SEAP reporter RAW -Blue cells (InvivoGen Cells, cat# raw-sp, San Diego CA) were grown in Dulbecco Modified Eagle Medium (DMEM) (Coming, Tewksbury MA) supplemented with 4.5% glucose, 10% fetal calf serum (FCS) (Coming), 2 mM L-glutamine, 1 x Penicillin-streptomycin (Gibco, Waltham MA), and 100 pg / mL zeocin (InvivoGen Cells) in a humidified incubator with 5% CO2 at 37°C. The cells were passaged when they reached 70% confluence for 6-10 generations. Cells were scraped, resuspended in fresh media, and plated in a flat-bottom 96-well tissue culture plate (Coming) at final density 2 x 105cells / well in 180 pL. The cells were incubated for 2 hours at 37°C in an atmosphere of 5% CO2, then treated with the target lipid compound at a final concentration from 0.1-125 pM in 1% or 2% (v / v) EtOH or with a blank reagent (EtOH) with final concentration 1% or 2% (v / v) EtOH as a control. After 1 hour of incubation, the lipid-cell co-cultures were treated with E. coli LPS (Sigma- Aldrich) to a final concentration of 10 pg / mL. After 24 hours of stimulation, supernatants were collected, and NFKB activation determined using the detection medium QUANTI-Blue, prepared according to the manufacturer’s recommendations.

[0151] Results

[0152] An experiment was performed to determine whether the administration of phosphatidylglycerol compounds affect innate immune responses in vitro. Prior results showed that early-life antibiotic exposure accelerated T1D onset by dysregulating genes involved in host intestinal innate immune responses (Zhang, X. S. et al., Cell Host Microbe 29, 1249-1265 el249, (2021) ). The compounds LPG(13:0), LPG(16:0), LPG(18:0) or PG(15:0_15:0) (Figure 6) were tested to determine whether they affect transcription of NFKB, the master regulator of innate immune responses (Dorrington, M. G. & Fraser, I. D. C., Front Immunol 10, 705, (2019); and Mussbacher, M., Derler, M., Basilio, J. & Schmid, J. A., Front Immunol 14, 1134661, (2023)). Incubating the phospholipids with mouse Raw Blue macrophage NFKB activity reporter cell lines in the absence of LPS stimulation, NFKB activity was unaltered (data not shown). However, when incubated with the Raw Blue cells in the presence of E. coli LPS, each phospholipid significantly suppressed the LPS-induced NFKB activity in a dose-dependent manner (Figure 1 A-D), consistent with anti-inflammatory effects through NFKB pathways. Under the same incubation conditions, retinoic acid, a known anti-inflammatory lipid (Sierra- Mondragon, E. et al., JNutr Biochem 60, 47-60, (2018)) significantly suppressed LPS- induced NFKB activity at concentrations as low as 5 pM. LPG(13:0) showed significant suppression also as low as 5 pM (Figure 1A), while the three other phospholipids showed significant suppression from 25 pM (Figure IB, C, D). Example 2. Administration of phosphatidylglycerol compounds to cultured human cells reduces expression of genes involved in inflammatory responses. (Figure 2).

[0153] Materials and Methods

[0154] Lipid compound standards. As in Example 1.

[0155] Preparation of lipids. As in Example 1

[0156] Cell culture and co-culture assays. Human colonic epithelial cell line HT-29 (ATCC, Manassas VA) was cultured in RPMI 1640 medium (Coming) with 10% FCS and lx penicillinstreptomycin in a humidified incubator with 5% CO2 at 37°C and passaged when they reached 90% confluence. For co-culturing with lipid compounds, the cells were scraped and resuspended in fresh media, and plated into 6-well tissue culture plates (Corning) and grown to >90% confluence in RPMI 1640 medium with 10% FBS without antibiotics (2 mL per well) and incubated for 1 hour to improve attachment, then each lipid compound added at a final concentration of 50 pM in 1% EtOH, or blank reagent EtOH as a reference control, incubating for 16 hours at 37°C in 5% CO2. The attached cells were washed with ice-cold PBS, resuspended in TRIzol reagent (Qiagen), and frozen at -80°C for RNA extraction.

[0157] RT-qPCR for host target gene expression. Total RNA was extracted from TRIzol-collected cells using the QIAgen Mini RNeasy kit (Qiagen) and cDNA was synthesized from the total RNA samples using the Verso cDNA kit (Thermo Scientific) according to the manufacturers' instructions. qPCR was performed on a LightCycler 480 system (Roche, Branchburg NJ) using 10 ng of synthesized cDNA, target gene-specific primer pairs (Table 1), and Power SYBR Green PCR Master mix (Roche). Target mRNA levels were normalized to 18S rRNA or the housekeeping gene GAPDH as internal controls for each sample. For group mean comparisons, the Mann-Whitney t-test was performed, with p-value < 0.05 indicating significance.

[0158] Table 1 DNA primers used for PCRs a Zhang, X. S. et al. , Cell Host Microbe 29, 1249-1265 el 249, (2021) b Lin, S. et al. Cancer Cell International 19, 282, (2019) c Wang, X. et al. Nucleic Acids Res 40, (2012) d Rho, H. W. et al. BMC Cancer 10, 240, (2010)

[0159] Results

[0160] The effect of phosphatidylglycerol compounds on innate responses in human gut epithelial cells, which directly interact with gut microbiota and microbially-produced lipids was investigated. Each compound was incubated with human colonic epithelial HT29 cells, a cell line grown in culture (Figure 2). PG(15:0_15:0) significantly repressed NFKB expression in HT29 cells. Both odd-chain compounds LPG(13:0) and PG(15:0_15:0) suppressed transcription factor RORyT, Toll-like receptor TLR5, and pro-inflammatory cytokine regulator Caspasel in the HT29 cells (Figure 2 A-D). LPG(13:0) also significantly suppressed the expression of IL- 13, while PG(15:0_15:0) significantly suppressed the expression of pro-inflammatory cytokine TNFa (Figure 2 A,B). The two even-chain LPGs, LPG(16:0) and LPG(18:0), increased NFKB expression in human HT29 cells (Figure 2 C,D). Similar to LPG(13:0) and PG(15:0_15:0), both LPG(16:0) and LPG(18:0) significantly decreased RORyT and TLR5 expression. The two evenchain compounds significantly suppressed mucin MUC2 and Intercellular Adhesion Molecule (ICAM) and induced expression of Endothelial Cell Adhesion Molecule (ECAM) in HT29 cells (Figure 2C,D). These differential observations, based on in vitro culture, indicate that the effects of these compounds on host innate responses are context-dependent.

[0161] Example 3. Administration of phosphatidylglycerol compounds to primary mouse small intestinal epithelial cells reduces expression of genes involved in inflammatory responses. (Figure 3).

[0162] Materials and Methods

[0163] Lipid compound standards. As in Example 1.

[0164] Preparation of lipids. As in Example 1

[0165] Cell culture and co-culture assays. As in Example 2.

[0166] Murine macrophage NFKB-SEAP reporter. As in Example 1

[0167] Human colonic epithelial cell line HT-29 (ATCC, Manassas VA). As in Example 2.

[0168] RT-qPCR for host target gene expression. As in Example 2

[0169] Mouse small intestinal epithelial cells (SIEC) were isolated as described (Sato, T. & Clevers, l., Methods Mol Biol 945, 319-328, (2013)) with the following modifications. Male NOD mice were sacrificed at P23, the small intestine was removed and opened longitudinally, rinsed with cold HBSS (Gibco) to remove contents, and cut into 2-cm pieces, and incubated on ice in 20 mL DMEM (Corning) supplemented with 10% FCS (Gibco) with Collagenase type I (Sigma-Aldrich) 200 U / mL at 37°C for 30 minutes, with gently shaking at 100 rpm. The epithelial layer was gently dissociated by pipetting up and down from intestinal tissue and collected, filtered through a 100 pm cell strainer (Fisher Sci, Waltham MA) and centrifuged at 300 g for 5 minutes to pellet cells. Cells pooled from 3 mice were cultured and passaged in DMEM medium (Corning) supplemented with 10% FCS and lx penicillin-streptomycin. For incubation with lipid compounds, fresh subcultured cells were seeded at high density into 12- well tissue culture plates (Corning) in DMEM medium with 10% FBS without antibiotics (1 mL per well), cultured to >90% confluence for 1 hour, then incubated in a 2 mL co-culture system for 16 hours at 37°C in 5% CO2 with each lipid compound (4 pL) at a final concentration of 50 pM in EtOH, or blank reagent EtOH as a reference control. The attached SIEC cells were washed with ice-cold PBS, resuspended in TRIzol and frozen at -80°C for RNA extraction.

[0170] Results

[0171] The effect of phosphatidylglycerol compounds on innate responses in mouse gut epithelial cells, which directly interact with gut microbiota and microbially-produced lipids was investigated. Each compound was incubated with isolated (primary) mouse small intestinal epithelial cells (SIEC) (Figure 3). LPG(13:0) significantly suppressed NFKB gene expression in the primary culture mouse SIEC. Both odd-chain compounds LPG(13:0) and PG(15:0_15:0) suppressed transcription factor RORyT, Toll-like receptor TLR5, and pro-inflammatory cytokine regulator Caspasel in the primary mouse SIEC (Figure 3A,B). No significant effects were observed for the two even-chain LPGs. These differential observations, based on in vitro culture (Examples 2 and 3), indicate that the effects of these compounds on host innate responses are cell-type- and context-dependent.

[0172] Example 4. Oral administration of phosphatidylglycerol compounds restores markers of intestinal immune function dysregulated by antibiotic exposure (Figure 4).

[0173] Materials and Methods

[0174] Lipid compound standards. As in Example 1.

[0175] Preparation of lipids. As in Example 1

[0176] Mice. The NOD / ShiLtJ Germ-free mice were obtained from Prof. Kathy D. McCoy laboratory of University of Calgary and C57BL / 6J Germ-free mice were purchased from Charles River Laboratories (Wilmington DE) and were bred and maintained in a gnotobiotic facility at Rutgers New Jersey Medical School with sterilized chow diets (LabDiet 5LG4, LabDiet, St. Louis MO). Conventional NOD / ShiLtJ mice and C57BL / 6J mice were purchased from Jackson Laboratory (Bar Harbor ME) and bred in an SPF vivarium at Rutgers University's School of Public Health animal facility and at the Robert Wood Johnson Medical School Research Tower animal facility, with chow diets (PicoLab 5058, LabDiet). Both germ-free and conventional mice (NOD / ShiLtJ and C57BL / 6J) in the above facilities were maintained in the same environment of 23 °C ± 1 °C, on a 12: 12-hour light-dark cycle. All animal procedures were approved by the Rutgers University Institutional Animal Care and Use Committee (IACUC protocols no. 201900013, 201900017 and 201900032).

[0177] Oral administration of lipids to mice (Figure 4). To evaluate the in vivo effects of administering specific lipid compounds to young NOD mice, dams and their litters were randomly assigned to control (C) or 1PAT antibiotic (IP) groups as previously described23. At postnatal day (P) 12, pups from 3 litters in the IP group received a single gavage of fresh prepared lipid compounds LPG(13:0), PG(15:0_15:0), LPG(16:0), or LPG(18:0), respectively, at a dose of 0.3 mg / kg body weight (60 pL) daily for one week to P18. As controls, pups in the C group received the same volume of the blank solvent reagent (PBS-2% EtOH). Additional litters of IP mice received: (i) cecal material transfer (CMT) from a pool of cecal contents from 3 male NOD mice at P23; or (ii) retinoic acid (3 mg / kg body weight) in a single daily dose from P12 to Pl 8. From each litter, four pups (2 male / 2 female) were sacrificed on Pl 9, and ileum and colon collected in RNAlater for RT-qPCR-based evaluation of expression of host genes associated with T1D development. Cecal contents were also collected and frozen for lipidomic analysis and 16S rRNA gene sequencing.

[0178] Results

[0179] The ability of phosphatidylglycerol compounds to counteract the effects of antibiotic- induced dysbiosis in mice was investigated (Figure 4). In NOD mice that had received early-life IP antibiotic-treatment, each compound was fed for 7 days to assess whether they could reverse the IP-induced alterations in early T1D marker genes in the ileum and the colon. Compared to control pups without antibiotic exposure, the IP treatment induced decreased ileal expression of REG3y and NOS2, two early biomarkers of T1D onset (Figure 5), consistent with prior findings (Zhang, X. S. et al., Elife 7, (2018); and Zhang, X. S. et al., Cell Elost Microbe 29, 1249- 1265 el249, (2021)). Cecal material transplant (CMT) from healthy mouse donors (Zhang, X. S. et al., Cell Elost Microbe 29, 1249-1265 el249, (2021)), used as a positive control, restored the decreased expression of REG3y and NOS2 induced by IP in the ileum, also as expected (Fig. 5). LPG(13:0), LPG(16:0), and PG(15:0_15:0) each significantly increased REG3y expression in the ileum although only partially restored expression compared to the control and CMT groups. Similarly, LPG(13:0), LPG(16:0), and PG(15:0_15:0) each significantly increased ileal NOS2 expression, but only LPG(16:0) fully restored expression to the level of the controls (Figure 5); in the colon, LPG(16:0) also significantly increased NOS2 gene expression compared to the IP- treated group (data not shown). These observations indicate that each of the phosphatidylglycerol compounds LPG(13:0), LPG(16:0), and PG(15:0_15:0) can restore the antibiotic-induced dysregulation. Administering LPG(18:0) group restored ileal REG3y and NOS2 expression to a minor extent without statistical significance (Figure 5). These studies provide evidence, consistent with in vitro data, that these microbial compounds are bioactive in the intestinal milieu and can be used to restore perturbed signaling after an antibiotic course.

[0180] Example 5. LPG16:0 and LPG18:0 alter mitochondrial respiration in intestinal epithelial cells (Figure 8).

[0181] Mitochondrial respiration measurement. The oxygen consumption rate (OCR) was measured using the Seahorse XF Cell Mito Stress Test Kit and the Seahorse XF Analyzer (Seahorse Bioscience, USA), following the manufacturer’s instructions. Briefly, fresh HT29 cells were seeded in XF96 microplates at a density of 1.25 * 105cells / mL per well in 180 pL of DMEM (Coming) supplemented with 10% FBS (Coming) and 10 mM glucose and incubated for 24 hours at 37°C in a humidified atmosphere containing 5% CO2. Cells were then treated for 24 h with the target lipid compound LPG16:0 at a final concentration of 0.5, 2, and 5 pM, or with LPG18:0 at 0.5, 1, and 2 pM, in 0.25% (v / v) PBS. A PBS-only treatment (0.25% v / v) served as the vehicle control. Prior to the assay, the Seahorse XF sensor cartridge was hydrated with XF calibrant (200 pL / well) and incubated overnight in a humidified, non-CCh 37°C incubator. The assay medium was prepared using XF DMEM (pH 7.4; Seahorse Bioscience) supplemented with 1 mM pyruvate, 2 mM glutamine, and 10 mM glucose. Cells were incubated and washed three times with assay medium. Three compounds or mixtures were sequentially injected into the microplate: oligomycin (1.0 pM) via Port A, carbonyl cyanide-4- (trifluoromethoxy)phenyl-hydrazone (FCCP; 2.0 pM) via Port B, and a mixture of rotenone and antimycin A (0.5 pM each) via Port C, in accordance with the XF Cell Mito Stress Test protocol (Hu, T. et al., Metabolism 146, 155658 (2023), doi.org: 10.1016 / j.metabol.2023.155658). After the assay, OCR values were normalized to total protein content per well, measured using the BCA assay, to account for differences in cell number. Protein-normalized values were used to calculate basal mitochondrial respiration, maximal respiration, ATP-linked oxygen consumption, and non-mitochondrial respiration.

[0182] Next, whether the broad effects from these compounds might reflect altering a fundamental characteristic of intestinal epithelial cells was investigated. Mitochondrial function was evaluated, since changing the homeostasis could have the observed downstream effects. First, the impact of LPG16:0 and LPG18:0 on HT29 intestinal epithelial cell oxygen consumption rate (OCR) was studied using the Seahorse XF Cell Mito Stress Test. (Figure 8A- F). LPG16:0 and LPG18:0 both significantly increased basal respiration compared to control (Figures 8B, 8G). Both compounds significantly increased maximal respiration and ATP- production in a dose-dependent manner (Figures 8C, 8D, 8H, 81). Similarly, compared to control, both LPG16:0 and LPG18:0 increased non-mitochondrial respiration of the HT29 cells in a dose-dependent pattern (Figures 8E, 8J). Coupling efficiency and spare respiratory capacity were not significantly affected by either lipid compound, while LPG18:0 tended to increase cell proton leak (data not shown). Parallel studies incubating HEK (non-intestinal) cells with LPG16:0 or 18:0 show effects similar to those in HT29 cells (data not shown). Altogether, these results indicate that LPG16:0 and LPG18:0 enhance multiple parameters of epithelial cell mitochondrial respiratory function, providing one mechanism for the altered physiology observed in vitro.

[0183] Example 6. Oral administration of LPG16:0 and LPG18:0 to NOD mice partially restores altered ileal gene expression and gut microbiome after antibiotic exposure.

[0184] To further investigate the mechanisms by which the lipid compounds LPG16:0 and LPG18:0 affect the host, intestinal gene expression in the antibiotic-treated NOD mice was examined. Specifically, control mice were compared with antibiotic (IPAT)-exposed mice that were given LPG16:0, LPG18:0, or vehicle control (PBS / EtOH). First, whether administering the LPGs affected the cecal and ileal microbiome was assessed. Usingl6S rRNA sequencing it was confirmed that the IP mice had significantly altered microbiome population structure, and partial restoration was found in the mice that had been administered LPG 16:0 or LPG 18:0. Next, microbial diversity and composition were evaluated in more detail. Cecal microbiome a- diversity, measured by the Shannon index and Pielou’s Evenness, was significantly reduced in IP-treated mice compared to Controls (p < 0.05), indicating reduced microbial richness and evenness following early-life antibiotic exposure. Notably, administration of LPG16:0 or LPG18:0 partially restored a-diversity in the cecum, so differences with control no longer were significant. MaAsLin2 analysis was performed to further identify taxa contributing to the observed differences. Several cecal taxa significantly altered by IP treatment were partially restored by either LPG16:0 or LPG18:0, including Lactobacillaceae g_HT002, which was consistently recovered after both lipid interventions. This suggests that specific microbial taxa may be selectively supported or re-established in response to these lipid compounds.

[0185] Microbiome composition in the ileum was also assessed. Principal coordinates analysis (PCoA) of Bray-Curtis P-diversity showed that antibiotic exposure significantly altered the microbial community structure in the ileum and that treatment with LPG16:0 or LPG18:0 led to microbial profiles that shifted closer to Control mice, indicating partial restoration. However, unlike in the cecum, ileal a-diversity metrics did not reveal significant differences across treatment groups, suggesting that the impact of lipid treatment on microbial richness and evenness may be more pronounced in the cecum than the ileum. Together, these findings demonstrate that administration of LPG16:0 and LPG18:0 may partially reverse antibiotic-induced disruptions in both the cecal and ileal microbiome.

[0186] Example 7. Oral administration of phosphatidylglycerols restores markers of intestinal immune function dysregulated by antibiotic exposure.

[0187] Oral administration of lipids to mice. To evaluate the in vivo effects of administering specific lipid compounds to young NOD mice, dams and their litters were randomly assigned to control (C) or 1PAT antibiotic (IP) groups as previously described (Zhang, X. S. et al., Cell Host Microbe 29, 1249-1265 el249 (2021), doi.org: 10.1016 / j.chom.2021.06.014). A therapeutic dose of the macrolide tylosin tartrate (Sigma-Aldrich, Billerica MA) was given to IP pups in their non-acidified drinking water at 333 mg / L on P5-P10. At postnatal day (P) 12, pups from 3 litters in the IP group received a single gavage of fresh prepared lipid compounds LPG(13:0), PG(15:0_15:0), LPG(16:0), or LPG(18:0), respectively, at a dose of 0.3 mg / kg body weight (60 pL) daily for one week to P18. As controls, pups in the C group received the same volume of the blank solvent reagent (PBS-2% EtOH). Additional litters of IP mice received: (i) cecal material transfer (CMT) from a pool of cecal contents from 3 male NOD mice at P23; or (ii) retinoic acid (3 mg / kg body weight) in a single daily dose from P12 to P18. From each litter, four pups (2 male / 2 female) were sacrificed on Pl 9, and ileum and colon collected in RNAlater for RT-qPCR-based evaluation of expression of host genes associated with T1D development. Cecal contents were also collected and frozen for lipidomic analysis and 16S rRNA gene sequencing.

[0188] RNA-seq and bioinformatics analysis. Total RNA was extracted from mouse ileal tissues using the RNeasy Plus Mini Kit (Qiagen) combined with on-column DNA digestion using the RNase-Free DNase Set (Qiagen). RNA quality and quantity were determined using NanoDrop (NanoDrop Technologies). Libraries were prepared with rRNA depletion method. Subsequent sequencing was performed on an Illumina platform (30 million paired-end reads per sample) with ERCC spike-in at Azenta Life Sciences (South Plainfield, NJ). Raw sequencing reads (fastq) were quality checked using fastQC (v.0.12.1) (Andrews, S., Babraham Bioinforma. 2010. Babraham Institute, Cambridge, United Kingdom (2020)). and aligned to mouse mm9 reference genome using Kallisto (v2.1.0) (Bray, N. L., Pimentel, H., Melsted, P. & Pachter, L., Nature biotechnology 34, 525-527 (2016)). Differential expression analysis was performed with DESeq2 (v.1.46.0; bioconductor.org / packages / DESeq2) (Love, M. I., Huber, W. & Anders, S., Genome biology 15, 1-21 (2014)) and KEGG pathway analyses performed with enrichKEGG package in the R interface (Yu, G., Wang, L. G., Han, Y. & He, Q. Y., Omics 16, 284-287 (2012), doi. org: 10.1089 / omi.2011.0118). All volcano plots were generated with the Enhanced Volcano package in the R interface (EnhancedVolcano. Publication-ready volcano plots with enhanced coloring and labeling (2024). bioconductor.org / packages / devel / bioc / vignettes / EnhancedVolcano / inst / doc / EnhancedVolcano.html). All heatmaps were generated with the pheatmap package (Pheatmap: Pretty heatmaps (2025). chrome-extension: / / efaidnbmnnnibpcajpcglclefindmkaj / https: / / cran. r-project.org / web / packages / pheatmap / pheatmap.pdf, using as the Euclidean distance metric for non-supervised hierarchical clustering.

[0189] Experiments were performed to assess whether directly administering phosphatidylglycerol compounds to mice could counteract the effects of the antibiotic-induced dysbiosis. NOD mice that had received early-life IP antibiotic-treatment were fed a single compound to assess whether they could reverse the IP-induced alterations in T1D marker ileal genes. Compared to control pups without antibiotic exposure, the IP treatment decreased ileal expression of REG3y and NOS2 (both p<0.01), two early biomarkers of T1D onset, consistent with our prior findings (Zhang, X. S. et al., Cell Host Microbe 29, 1249-1265 el249 (2021), doi.org: 10.1016 / j.chom.2021.06.014; and Zhang, X. S. et al., Elife 7 (2018). doi.org: 10.7554 / eLife.37816). CMT from healthy mouse donors restored the decreased expression of REG3y and NOS2 induced by IP in the ileum. Both LPG(16:0) and LPG(18:0) also increased REG3y and NOS2 expression in the ileum (significant reduction seen with IP alone compared with Control now also lost). Consistent with the in vitro data, these studies provided preliminary in vivo evidence of the bioactivity of these microbial compounds in the intestinal milieu and their potential to restore perturbed signaling after an antibiotic course.

[0190] Oral administration of LPG16:0 and LPG18:0 partially restored the ileal gene expression profile altered by antibiotic exposure. To further assess the effects of LPG16:0 and LPG18:0 administration on ileal gene expression profile of 1PAT mice at a transcriptomic level [in addition to individual gene levels in the above text], bulk RNA-seq was performed on ileal tissues collected at P19 from antibiotic-treated male NOD mice with or without a period of 7- day lipid administration, as well as control mice without antibiotic exposure. 21,924 transcripts were identified for differentially expressed gene (DEG) analysis. Both of the groups to which LPG16:0 or LPG18:0 were given after the 1PAT treatment clustered closer to controls (adjusted p value =0.095 for both), suggesting administration of these lipid compounds partially restored gene expression profiles, without significant differences between the two LPG groups (adjusted p value = 0.69). KEGG pathway enrichment analysis showed that giving either LPG16:0 or LPG18:0 restored 1PAT mouse ileal vitamin digestion and absorption, protein digestion and absorption, primary bile acid biosynthesis, fatty acid metabolism and neuroactive ligandreceptor interaction pathways (Figure 9A and 10). Both LPG16:0 and LPG18:0 decreased 1PAT mice ileal proteasome pathway and phagosome pathway (Figure 9A and 10, Table 2), which may suggest that administration of these two lipids may improve ileal barrier integrity or mucosal immunity and relieve ileal anti-invasion burden. The 1PAT mice showed a large number of significantly up- and downregulated genes (total of 307 DEGs) compared with the control mice reflecting the broad impact of early-life antibiotic exposure (Figure 9B, left panel). In contrast, administering LPG16:0 or LPG18:0 after the 1PAT exposure resulted in much smaller numbers of significantly deviated DEGs vs Control (36 for LPG16:0 and 49 for LPG18:0) (Figure 9B, middle and right panels), consistent with partial normalization of gene expression. In a Venn diagram based on the DEGs from IP-, LPG16:0-, and LPG18:0-treated groups (Figure 9C), 270 genes were uniquely altered in 1PAT mice, with 139 upregulated and 131 downregulated compared with the Controls (Group I) (Figure 9C). Both LPG16:0 and LPG18:0 similarly and partially restored the effects of the antibiotic exposure. Unsupervised hierarchical clustering of the 270 Group I genes (representing transcriptional changes induced by antibiotic exposure) revealed three distinct clusters (data not shown). This clustering pattern indicates clear transcriptomic differences between IP and control mice, with partial restoration of the control-like gene expression profile by LPG16:0 and LPG18:0 administration. Further analysis identified a subset of 131 genes that were upregulated in the control -like cluster compared to the 1PAT clusters, and a subset of 44 genes that were downregulated in the controllike cluster (data not shown). KEGG analysis of this gene set revealed pathways involved in epithelial barrier function, lipid metabolism, and immune regulation that were disrupted by antibiotics and normalized by lipid administration (Figure 9D). Representative genes affected by treatment include Trim40, Sprr2al, and Socsl, all significantly upregulated by IP and returned to baseline with either lipid administration (Figure 10). In contrast, the long noncoding transcript A930038B10Rik was downregulated by antibiotics and trended toward restoration with LPG16:0 or LPG18:0 (Figure 10). Altogether, these results demonstrate that both phospholipid treatments can partially reverse antibiotic-induced transcriptional perturbations in the ileum. Table 2. List of 23 Group I genes (of 270) associated with lysosome or mitochondria.

[0191] Gene Biological function Associated organelle

[0192] Anxal3 Annexin; vesicle trafficking Lysosome

[0193] Fabp2 Fatty acid-binding protein, lipid transport Lysosome

[0194] Flvcrl Iron transporter Lysosome

[0195] Rabllfip Vesicle trafficking adaptor Lysosome

[0196] 5 Samd8 Sphingomyelin synthase Lysosome

[0197] Tmeml44 Membrane protein, lysosome associated Lysosome

[0198] Gpx2 Glutathione peroxidase Lysosome

[0199] Marchf3 E3 ubiquitin ligase Lysosome

[0200] Acox2 Fatty acid -oxidation enzyme Mitochondria

[0201] Acsl5 Long-chain acyl-CoA synthetase Mitochondria

[0202] Efhdl Calcium-binding mitochondrial protein Mitochondria

[0203] Gludl Glutamate dehydrogenase Mitochondria

[0204] Maob Monoamine oxidase B Mitochondria

[0205] Mtl Metallothionein; stress response Mitochondria

[0206] Ppargcla Transcriptional coactivator for mitochondrial Mitochondria biogenesis

[0207] Abcd2 Peroxisomal transporter, lipid metabolism Mitochondria

[0208] Dmgdh Dimethylglycine dehydrogenase Mitochondria

[0209] Gsr Glutathione reductase Mitochondria

[0210] Hspb2 Heat shock protein; mitochondrial maintenance Mitochondria

[0211] Hspb6 Heat shock protein; mitochondrial protection Mitochondria

[0212] Nnmt Nicotinamide N-methyltransferase Mitochondria

[0213] Nos2 Nitric oxide synthase Mitochondria

[0214] Nlrxl NOD-like receptor in mitochondria, immune Mitochondria & signaling Lysosome

[0215] Genes shown in bold were downregulated in 1PAT (vs. Control). and restored by both LPG16:0 and LPG18:0. Genes shown in gray were upregulated in 1PAT (vs. Control) and restored by both LPG16:0 and LPG18:0.

[0216] Discussion

[0217] Prior studies of NOD mice showed that antibiotic exposure induced gut microbiome perturbation in early life which accelerated T1D onset, through host innate immune responses (Zhang, X. S. etal., Cell Host Microbe 29, 1249-1265 el 249 (2021), doi. org: 10.1016 / j.chom.2021.06.014; Zhang, X. S. et al., Elife 7 (2018). doi.org: 10.7554 / eLife.37816; and Livanos, A. E. etal., Nat Microbiol 1, 16140 (2016). doi.org: 10.1038 / nmicrobiol.2016.140) and that this could be restored by maternal cecal microbiota transplant (CMT). Based on metagenomic analyses of cecal microbiome, which highlights differentiation of bacterial lipid metabolism pathways during microbiota perturbation, gut microbiota lipid profiles have been systematically characterized in conventional and germ- free mice, identifying a group of specific microbiota-produced lipid compounds. Using a pipeline comparing lipidomics of gut microbiota from antibiotic-treated mice and humans and their untreated controls as a strategy for discovery, from >700 identified lipids, a group of gut bacterial-derived lipids were identified that had been reduced by the antibiotic treatments, and at least partially restored by the CMT. Then a subset of these compounds with defined structures was evaluated to further investigate their biological activities in vitro and in vivo.

[0218] Bacterial-derived lipids include highly diverse and abundant phospholipids such as LPG, PG, LPC, PC, and CL, released from bacterial cell membranes or outer membrane vesicles (OMVs) and could be critical in interacting with host immune mechanisms (Brown, E. M., Clardy, J. & Xavier, R. J., Cell Host Microbe 31, 173-186 (2023). doi. org: 10.1016 / j.chom.2023.01.009; Ryan, E., Joyce, S. A. & Clarke, D. J., Microbiology (Reading) 169 (2023). doi. org: 10.1099 / mic.0.001315; Schoeler, M. & Caesar, R., Rev Endocr Metab Disord 20, 461-472 (2019). doi.org: 10.1007 / sl l l54-019-09512-0; Lin, D. L. et al., J Immunol Res 2024, 2506586 (2024). doi. org:10.1155 / 2024 / 2506586; and Joyce, L. R. & Doran, K. S., PLoS Pathog 19, elOl 1026 (2023). doi.org: 10.1371 / journal.ppat.1011026). The structural diversity of phospholipids, reflecting the biochemical and biophysical characteristics of bacterial membranes that enable bacterial adaptations in the complex gut environment, also could differentially mediate host-microbe communication and modify host immune responses (Brown, E. M., Clardy, J. & Xavier, R. J., Cell Host Microbe 31, 173-186 (2023), doi. org: 10.1016 / j.chom.2023.01.009; Ryan, E., Joyce, S. A. & Clarke, D. J., Microbiology (Reading) 169 (2023), doi.org: 10.1099 / mic.0.001315; van Meer, G., Voelker, D. R. & Feigenson, G. W ., Nat Rev Mol Cell Biol 9, 112-124 (2008), doi.org: 10.1038 / nrm2330; and Sohlenkamp, C. & Geiger, O., FEMS Microbiol Rev 40, 133-159 (2016), doi.org: 10.1093 / femsre / fuv008). Four defined phospholipids, LPG(13:0), LPG(16:0), LPG(18:0), and PG(15:0_15:0) whose abundance consistently varied between microbiota samples from antibiotic-treated and control subjects, were intensively studied as representing the potential of the broader group for host interactions. The results suggested that these bacterial lipids could serve as therapeutic agents for the prevention or treatment of inflammatory and immune-mediated processes originating in the gut, including T1D and IBD (Fenneman, A. C., Weidner, M., Chen, L. A., Nieuwdorp, M. & Blaser, M. J., Nat Rev Gastroenterol Hepatol 20, 81-100 (2023), doi.org: 10.1038 / s41575-022-00685-9).

[0219] Mitochondria play essential roles in immune cell signaling, energy metabolism, and regulation of inflammatory responses by their multiple energy-generating biochemical functions (Angajala, A. et al., Front Immunol 9, 1605 (2018). doi. org: 10.3389 / fimmu.2018.01605). It has now been shown that specific lipid compounds, LPG16:0 and LPG18:0, directly modulate mitochondrial function in intestinal epithelial cells. These findings align with recent reports that phospholipids, sphingolipids, and sterols are essential for the proper assembly and activity of electron transport chain complexes, thereby affecting energy production, membrane potential, and cellular homeostasis (Martensson, C. U., Doan, K. N. & Becker, T., Biochim Biophys Acta Mol Cell Biol Lipids 1862, 102-113 (2017), doi.org: 10.1016 / j.bbalip.2016.06.015; and Nielson, J. R. & Rutter, J. P., J Biol Chem 293, 7517-7521 (2018), doi.org: 10.1074 / jbc.Rl 17.001655). These results add a new dimension to the role of gut bacteria-derived lipids: beyond serving as structural or metabolic intermediates, they may function as regulators of host mitochondrial activity with downstream effects on epithelial defense and immune homeostasis. Recent findings show that serum LPG18:0 is elevated in asthma patients and impairs the differentiation of regulatory T cells (Tregs) from naive CD4+T cells (Aili, A. et al., T-cells. Eur Respir J 64 (2024), doi.org: 10.1183 / 13993003.01752-2023). The findings herein indicate that the roles of these biologically-active compounds are context-dependent and may vary by tissue and cell type.

[0220] Example 8. The following illustrate representative pharmaceutical dosage forms, containing a compound of formula (I) ('Compound X'), for therapeutic or prophylactic use in humans.

[0221] (i) Tablet 1 mg / tablet

[0222] Compound X= 100.0

[0223] Lactose 77.5

[0224] Povidone 15.0

[0225] Croscarmellose sodium 12.0

[0226] Microcrystalline cellulose 92.5

[0227] Magnesium stearate 3,0

[0228] 300.0

[0229] (ii) Tablet 2 mg / tablet

[0230] Compound X= 20.0

[0231] Microcrystalline cellulose 410.0

[0232] Starch 50.0

[0233] Sodium starch glycolate 15.0

[0234] Magnesium stearate 5,0

[0235] 500.0 (iii) Capsule mg / capsule

[0236] Compound X= 10.0

[0237] Colloidal silicon dioxide 1.5

[0238] Lactose 465.5

[0239] Pregelatinized starch 120.0

[0240] Magnesium stearate 3,0

[0241] 600.0

[0242] (iv) Injection 1 (1 mg / ml) mg / ml

[0243] Compound X= (free acid form) 1.0

[0244] Dibasic sodium phosphate 12.0

[0245] Monobasic sodium phosphate 0.7

[0246] Sodium chloride 4.5

[0247] 1.0 N Sodium hydroxide solution

[0248] (pH adjustment to 7.0-7.5) q.s.

[0249] Water for injection q.s. ad 1 mL

[0250] (v) Injection 2 (10 mg / ml) mg / ml

[0251] Compound X= (free acid form) 10.0

[0252] Monobasic sodium phosphate 0.3

[0253] Dibasic sodium phosphate 1.1

[0254] Polyethylene glycol 400 200.0

[0255] 1.0 N Sodium hydroxide solution (pH adjustment to 7.0-7.5) q.s.

[0256] Water for injection q.s. ad 1 mL

[0257] (vi) Aerosol mg / can

[0258] Compound X= 20.0

[0259] Oleic acid 10.0

[0260] Tri chi oromonofluorom ethane 5,000.0

[0261] Dichlorodifluoromethane 10,000.0

[0262] Dichlorotetrafluoroethane 5,000.0

[0263] The above formulations may be obtained by conventional procedures well-known in the pharmaceutical art. All publications, patents, and patent documents are incorporated by reference herein, as though individually incorporated by reference. The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

Claims

CLAIMSWhat is claimed is:

1. A method comprising, modulating intestinal immune function in an animal or modulating inflammatory responses in the gut of an animal, by modulating the amount of one or more phosphatidylglycerol compounds in the animal.

2. The method of claim 1, which is a method for modulating intestinal immune function in an animal.

3. The method of claim 1, which is a method for modulating inflammatory responses in the gut of an animal.

4. The method of claim 2, which is a method for restoring intestinal immune function in an animal following antibiotic treatment.

5. The method of claim 3, which is a method to reduce inflammatory response in the gut.

6. The method of claim 5, wherein the animal has an increased immune response in the gut associated with a disease selected from the group consisting of Type 1 diabetes, inflammatory bowel diseases (including, Crohn’s disease, Ulcerative colitis, and Celiac disease); asthma, eczema, food allergies, allergic rhinitis (hay fever), psoriasis, type 2 diabetes, obesity, atherosclerotic cardiovascular disease (ASCVD), Parkinson’s disease, Alzheimer’s disease, and multiple sclerosis.

7. A method comprising, treating an inflammatory disorder, autoimmune disorder, an allergic disorder, and / or a degenerative disorder in an animal, by modulating the amount of one or more phosphatidylglycerol compounds in the animal.

8. The method of claim 7, wherein the inflammatory, autoimmune, allergic, or degenerative disorder is associated with antibiotic treatment.

9. A method comprising, restoring the integrity of gut epithelium in an animal following antibiotic treatment, by modulating the amount of one or more phosphatidylglycerol compounds in the animal.

10. A method comprising improving mitochondrial function in epithelial cells in an animal by modulating the amount of one or more phosphatidylglycerol compounds in the animal.

11. A method comprising, restoring gene expression in an animal in which gene expression has been altered, by modulating the amount of one or more phosphatidylglycerol compounds in the animal.

12. The method of claim 11 wherein the gene expression has been altered by an inflammatory process, antibiotic treatment, or cancer.

13. A method comprising, restoring gene expression in an animal in which gene expression of the gut epithelium has been altered by antibiotic treatment, by modulating the amount of one or more phosphatidylglycerol compounds in the animal.

14. A method comprising, mitigating tissue injury in an animal, by modulating the amount of one or more phosphatidylglycerol compounds in the animal.

15. The method of claim 14, wherein the tissue injury is associated with an inflammatory process, antibiotic treatment, or cancer.

16. The method of any one of claims 1-15, wherein the amount of one or more phosphatidylglycerol compounds is modulated in the animal by administering a phosphatidylglycerol compound to the animal.

17. The method of claim 16, wherein the phosphatidylglycerol compound is a compound of formula (I):or a salt thereof, wherein:R1is a saturated or unsaturated, branched or straight hydrocarbon chain comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, which is optionally substituted with one or more halo groups;R2is H or a saturated or unsaturated, branched or straight hydrocarbon chain comprising12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms, which is optionally substituted with one or more halo groups;R3is OH, (Ci-Ce)alkoxy, (Ci-C6)cycloalkyloxy, or (Ci-Ce)alkanoyloxy, which (Ci- Ce)alkoxy, (Ci-C6)cycloalkyloxy, and (Ci-Ce)alkanoyloxy is optionally substituted with one or more groups independently selected from the group consisting of halo hydroxy, -N+(Ra)(Rb)(Rc)X' , and (Ci-C3)alkoxy;Rais (Ci-C6)alkyl;Rbis (Ci-C6)alkyl;Rcis (Ci-Ce)alkyl; andX is a suitable counter-anion.

18. The method of claim 17, wherein R1is a saturated hydrocarbon chain comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.

19. The method of claim 17, wherein R1is an unsaturated hydrocarbon chain comprising 12,13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.

20. The method of claim 19, wherein R1comprises one or two double bonds.

21. The method of claim 19, wherein R1comprises one double bond.

22. The method of claim 19, wherein R1comprises one or two triple bonds.

23. The method of claim 19, wherein R1comprises one triple bond.

24. The method of any one of claims 17-23, wherein R1is a branched hydrocarbon chain.

25. The method of any one of claims 17-23, wherein R1is a straight hydrocarbon chain.

26. The method of any one of claims 17-25, wherein R1comprises 13, 15, 17, or 19 carbon atoms.

27. The method of any one of claims 17-25, wherein R1comprises 12, 14, 16, 18, or 20 carbon atoms.

28. The method of any one of claims 17-27, wherein R2is a saturated hydrocarbon chain comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.

29. The method of any one of claims 17-27, wherein R2is an unsaturated hydrocarbon chain comprising 12, 13, 14, 15, 16, 17, 18, 19, or 20 carbon atoms.

30. The method of claim 29, wherein R2comprises one or two double bonds.

31. The method of claim 29, wherein R2comprises one double bond.

32. The method of claim 29, wherein R2comprises one or two triple bonds.

33. The method of claim 29, wherein R2comprises one triple bond.

34. The method of any one of claims 17-33, wherein R2is a branched hydrocarbon chain.

35. The method of any one of claims 17-33, wherein R2is a straight hydrocarbon chain.

36. The method of any one of claims 17-35, wherein R2comprises 13, 15, 17, or 19 carbon atoms.

37. The method of any one of claims 17-35, wherein R2comprises 12, 14, 16, 18, or 20 carbon atoms.

38. The method of any one of claims 17-35, wherein R2is H.

39. The method of any one of claims 17-38, wherein R3is OH.

40. The method of any one of claims 17-38, wherein R3is (Ci-Ce)alkoxy that is optionally substituted with one or more groups independently selected from the group consisting of halo hydroxy, and (Ci-C3)alkoxy.

41. The method of any one of claims 17-38, wherein R3is (Ci-Ce)alkoxy that is optionally substituted with one or more groups independently selected from the group consisting of halo hydroxy, and (Ci-C3)alkoxy.

42. The method of any one of claims 17-38, wherein R3is (Ci-Ce)alkoxy that is substituted with one or more hydroxy groups.

43. The method of any one of claims 17-38, wherein R3is 2,3-dihydroxypropoxy.

44. The method of any one of claims 17-38, wherein R3is (Ci-Ce)alkanoyloxy that is optionally substituted with one or more groups independently selected from the group consisting of halo hydroxy, and (Ci-C3)alkoxy.

45. The method of any one of claims 17-38, wherein R3is (Ci-Ce)alkoxy that is optionally substituted with -N+(Ra)(Rb)(Rc)X'; wherein Rais methyl; Rbis methyl; and Rcis methyl.

46. The method of any one of claims 17-38, wherein R3is 2,3-dihydroxypropoxy, 2, 3, 4, 5, 6-pentahydroxycyclohexyloxy, or 2-(trimethylammonium chloride)ethoxy.

47. The method of any one of claims 17-38 and 45, wherein X is chloro or bromo.

48. The method of any one of claims 1-16, wherein the phosphatidylglycerol compound is LPG( 13:0), LPG( 16 : 0), or PG( 15 : 0 15 : 0) or a salt thereof.

49. A pharmaceutical composition comprising a phosphatidylglycerol compound, for modulating intestinal immune function in an animal, modulating an inflammatory response in the gut of an animal, treating an inflammatory disorder, treating autoimmune disorder, treating an allergic disorder, and / or treating a degenerative disorder in an animal.

50. The pharmaceutical composition of claim 49, wherein the phosphatidylglycerol compound is a compound of formula (I) or a salt thereof as described in any one of claims 17-47.

51. The pharmaceutical composition of claim 49 or 50, wherein the animal is a human.

52. The pharmaceutical composition of claim 51, wherein the human has previously been treated with an antibiotic.

53. A phosphatidylglycerol compound for use in medical therapy.

54. The phosphatidylglycerol compound of claim 53, which is a compound, or a salt as described in any one of claims 17-48.

55. A phosphatidylglycerol compound for modulating intestinal immune function or modulating inflammatory response.

56. A phosphatidylglycerol compound for the prophylactic or therapeutic treatment of an inflammatory disorder, an autoimmune disorder, an allergic disorder, or a degenerative disorder.

57. A phosphatidylglycerol compound for restoring integrity of gut epithelium in an animal following antibiotic treatment.

58. A phosphatidylglycerol compound for improving mitochondrial function in epithelial cells.

59. A phosphatidylglycerol compound for restoring gene expression in an animal in which gene expression has been altered.

60. The phosphatidylglycerol compound of claim 59 wherein the gene expression has been altered by an inflammatory process, antibiotic treatment, or cancer.

61. A phosphatidylglycerol compound for restoring gene expression of the gut epithelium in an animal in which gene expression of the gut epithelium has been altered by antibiotic treatment.

62. A phosphatidylglycerol compound for mitigating tissue injury in an animal.

63. The phosphatidylglycerol compound of claim 62 wherein the tissue injury is associated with an inflammatory process, antibiotic treatment, or cancer.

64. The phosphatidylglycerol compound of any one of claims 55-63, which is a compound, or a salt as described in any one of claims 17-48.

65. Use of a phosphatidylglycerol compound to prepare a medicament for modulating intestinal immune function or modulating inflammatory response in an animal.

66. Use of a phosphatidylglycerol compound to prepare a medicament for treating an inflammatory disorder, an autoimmune disorder, an allergic disorder, or a degenerative disorder in an animal.

67. Use of a phosphatidylglycerol compound to prepare a medicament for restoring integrity of gut epithelium in an animal following antibiotic treatment.

68. Use of a phosphatidylglycerol compound to prepare a medicament for improving mitochondrial function in epithelial cells in an animal.

69. Use of a phosphatidylglycerol compound to prepare a medicament for restoring gene expression in an animal in which gene expression has been altered.

70. The use of claim 69, wherein gene expression in the animal has been altered by an inflammatory process, antibiotic treatment, or cancer.

71. Use of a phosphatidylglycerol compound to prepare a medicament for restoring gene expression of the gut epithelium in an animal in which gene expression of the gut epithelium has been altered by antibiotic treatment.

72. Use of a phosphatidylglycerol compound to prepare a medicament for mitigating tissue injury in an animal.

73. The use of claim 72 wherein the tissue injury is associated with an inflammatory process, antibiotic treatment, or cancer.

74. The use of any one of claims 65-73, wherein the phosphatidylglycerol compound is a compound or a salt as described in any one of claims 17-48.

75. A method comprising, restoring a level of a phosphatidylglycerol compound in an animal, by administering the phosphatidylglycerol compound to the animal.

76. A phosphatidylglycerol compound for restoring a level of a phosphatidylglycerol compound.

77. The use of a phosphatidylglycerol compound to prepare a medicament for restoring a level of the phosphatidylglycerol compound in an animal.

78. The method of claim 75, the compound of claim 76, or the use of claim 77, wherein the level of the phosphatidylglycerol compound has been reduced by an inflammatory process, antibiotic treatment, or cancer.

79. Any one of claims 75-78, wherein the phosphatidylglycerol compound is a compound or a salt as described in any one of claims 17-48.

80. The method of any one of claims 1-48, wherein the amount of one or more phosphatidylglycerol compounds is modulated in the animal by administering a unit dosage form that comprises the phosphatidylglycerol compound to the animal.

81. The method of any one of claims 75, 78, or 79, wherein a unit dosage form comprising the phosphatidylglycerol compound is administered.

82. The pharmaceutical composition of any one of claims 49-52 that is formulated as a unit dosage form.

83. The phosphatidylglycerol compound of any one of claims 53-64, 76, 78, and 79 that is formulated as a unit dosage form.

84. The use of any one of claims 65-74 and 77-79, wherein the medicament is a unit dosage form comprising the phosphatidylglycerol compound.

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